Leucine for use in the treatment of cancer

Abstract

The invention relates to leucine as food supplement in the treatment of cancer In particular leucine, when used as food supplement, enhances the effects of cycling fasting, such as the effects of a Fasting Mimicking Diet (FMD), and / or immune checkpoint inhibitors (ICIs), chemotherapy, chemotherapy plus immune checkpoint inhibitors, alone or in combination with fasting / FMD.

Description

[0001] LEUCINE FOR USE IN THE TREATMENT OF CANCER

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to leucine as food supplement in the treatment of cancer. In particular leucine, when used as food supplement, enhances the effects of cycling fasting, such as the effects of a Fasting Mimicking Diet (FMD), and / or immune checkpoint inhibitors (ICIs), chemotherapy, chemotherapy plus immune checkpoint inhibitors, alone or in combination with fasting / FMD.

[0004] BACKGROUND OF THE INVENTION

[0005] Breast cancer and lung cancer are the main leading cause of cancer death in women (17%) and men (27%) worldwide, respectively1. In both sexes, colorectal cancer is the second leading cause of cancer death (11% among men and 12% among women). These tumors are associated with low 5- year survival rates when diagnosed in the metastatic stage1. For this reason, lung, breast and colorectal cancer are considered the "three big killers" among human neoplasms, and are therefore the subject of many pre-clinical and clinical studies aimed at identifying new, effective, safe and economically sustainable therapies to prolong patient survival and, potentially, to increase tumor cure rates. In fact, despite recent therapeutic advances, which include new combinations of chemotherapy drugs, or the combination of chemotherapy with immunotherapy, these tumors are still associated with relatively high post-surgical recurrence rates and poor long-term outcomes1. This highlights the necessity to find new strategies to improve the efficacy of chemoimmunotherapies regimens in patients with these tumor types.

[0006] Recent preclinical studies have shown that specific experimental nutritional interventions, such as cyclic fasting or fasting-mimicking diets (FMD), can produce additive or synergistic anticancer effects when combined with standard chemotherapy or immunotherapy in several syngeneic mouse models, including triple negative breast cancer, colorectal and lung cancer models2-7. In detail, water-only fasting refers to cyclic and total avoidance of calorie intake for a certain amount of consecutive hours or days, whereas the term FMD refers to an heterogeneous group of cyclic, calorie-restricted, low-carbohydrate, low-protein nutritional interventions that aim at producing the same biological, metabolic and antitumor effects of cyclic water only fasting.23'25In this respect, water-only fasting can be considered the extreme part of the FMD spectrum, with preclinical data showing that these two approaches are equivalent in terms of immunomodulatory and antitumor activity.2In mice, water-only fasting and FMD produce similar and desirable immunomodulatory modifications, including an increase of tumor-infiltrating cytotoxic CD8+ T cells and a reduction of immunosuppressive regulatory T cells (Tregs) in syngeneic TNBC and lung mouse cancer models2,4.

[0007] A recent clinical trial conducted by the research group of present inventors in 101 cancer patients treated with standard therapies in combination with a five-day cyclic FMD regimen has shown that cyclic FMD is associated with positive immunomodulatory effects, leading to the downregulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) at both systemic and tumor levels, paralleled by a boost of the antitumor immune compartment, including an increase in cytotoxic T lymphocytes, natural killer (NK) cells and T lymphocytes with memory phenotype8. In addition, some patients with metastatic TNBC, CRC or lung cancer enrolled in this trial, and undergoing cyclic FMD in combination with standard chemotherapy or immunotherapy, achieved complete and long-lasting tumor remissions9,24. More recently, the research group of the present inventors also showed that cyclic FMD in combination with preoperative chemotherapy is associated with excellent tolerability and promising antitumor activity and efficacy in 30 women with localized triple-negative breast carcinoma (TNBC).24Based on these preclinical and clinical results, cyclic FMD is emerging as a promising antineoplastic nutritional intervention that could boost the anticancer activity of conventional anticancer treatments.23Understanding the molecular mechanisms underlying FMD-induced immunomodulatory activity will help in the development of new strategies to enhance the immunomodulatory and, potentially, antitumor effects of cyclic FMD, either alone or in combination with immune checkpoint inhibitors (ICIs), chemotherapy, or a combination of chemotherapy and ICIs.

[0008] SUMMARY OF THE INVENTION

[0009] In the present invention, the inventors demonstrated that cyclic water-only fasting in mice is associated with specific metabolic changes, including the modulation of systemic and intratumor metabolism of branched chain amino acids (BCAAs), namely leucine, isoleucine and valine (BCAA). Starting from this finding, they investigated whether fasting / FMD-induced increase of blood BCAA levels could contribute to the immunomodulatory and antitumor effects of nutrient starvation. For instance, leucine is a potent activator of mTORCl10 11, which plays a crucial role in T lymphocyte activation and proliferation in response to antigen exposure. The present invention demonstrates that oral leucine supplementation delays tumor progression and improves the survival of tumor-bearing mice. In addition, oral leucine supplementaton significantly potentiates fasting / FMD-induced immunomodulatory and anticancer effects, and this synergistic antitumor effects could derive from the cooperation of oral leucine supplementation and fasting / FMD in increasing blood leucine concentration. Finally, oral leucine supplementation, both alone and in combination with fasting / FMD, potentiates the efficacy of ICIs, chemotherapy, or combinations of chemotherapy and ICIs in the treatment of these highly aggressive tumor types.

[0010] Therefore the combination of leucine supplementation with fasting / FMD and / or with an optimized chemotherapy, immunotherapy or chemoimmunotherapy treatment is particularly advantageous in that it enhances the anti-cancer effect of cyclic water-only fasting or FMD, either alone or in combination with chemotherapy, immunotherapy or chemoimmunotherapy. Specifically, the combination of the invention is able to delay tumor progression given that leucine, when used as food supplement, potentiates the efficacy of other therapeutic interventions, such as chemotherapy, immunotherapy or chemoimmunotherapy combinations, and it also leads to increased cancer cure rates in some models of breast cancer and colorectal cancer. The combination of the invention is particularly advantageous also because it is safe and very well tolerated, with no toxicities emerging when oral leucine is combined with water-only fasting, chemotherapy, chemotherapy plus fasting, immunotherapy, immunotherapy plus fasting, chemoimmunotherapy, chemoimmunotherapy plus fasting. The object of the present invention is the use of oral leucine supplementation as an umbrella anticancer therapy that could be implemented in several clinical contexts in patients with different tumor types and receiving different standard-of-care concomitant therapies.

[0011] It is therefore an object of the invention leucine or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, either used alone or in combination with at least one cycle of water- only fasting or fasting mimicking diet (FMD) and / or a further therapeutic interventions.

[0012] Preferably said at least one water-only fasting cycle is characterized by 24 h to 72 h, preferably 24 h to 48 h, more preferably 48 h of water-only fasting 100% caloric restriction.

[0013] Still preferably said fasting mimicking diet lasts for a period of 24 to 190 hours, preferably said fasting mimicking diet lasts for a period of 24 to 150 hours, preferably said fasting mimicking diet lasts for approximately 120 hours.

[0014] In a further preferred embodiment of the invention said fasting mimicking diet is a regular caloric intake reduced by 65% to 85% and / or said fasting mimicking diet is based on an average caloric intake of between 300-600 Kcal / day.

[0015] Preferably the fasting mimicking diet comprises a first period of 0 to 24 hours wherein the caloric intake is a regular caloric intake reduced by approximately 65%, followed by a second period of 24 to 96 hours wherein the caloric intake is a regular caloric intake reduced by approximately 85%, preferably the first period lasts approximately 24 hours and the second period lasts approximately between 48 and 96 hours.

[0016] In a further preferred embodiment of the invention said fasting mimicking diet comprises a reduced protein intake and / or a reduced simple carbohydrate intake, and / or a relatively increased content of unsaturated fat intake.

[0017] Still preferably said fasting mimicking diet comprises administering a food having a relative content of monounsaturated and / or polyunsaturated fats between 10 % and 80 % ot the total calorie amount, a content of proteins from 0 to 10% of the total calorie amount, and a content of carbohydrates from 5% to 50% of the total calorie amount.

[0018] It is a further object of the invention leucine or the pharmaceutically acceptable salt thereof for use as defined above , wherein said leucine is administered in an amount of approximately from between lOOmg / kg and lOOOmg / kg on a daily basis, preferably between 150 mg / kg and 800mg / kg on a daily basis, preferably between 250 mg / kg and 600 mg / kg on a daily basis.

[0019] It is a further object of the invention or the pharmaceutically acceptable salt thereof for use as defined above wherein leucine is administered in an amount of approximately between 10 mg / kg and 1000 mg / kg on a daily basis, preferably between 25 mg / kg and 800 mg / kg on a daily basis, preferably between 50 mg / kg and 500 mg / kg on a daily basis.

[0020] It is a further object of the invention leucine or the pharmaceutically acceptable salt thereof for use as defined above and / or in combination with a further therapeutic intervention selected from the group consisting of: surgery, radiotherapy and a further therapeutic agent.

[0021] Preferably said further therapeutic agent is an immune checkpoint inhibitor (ICI), preferably a PD- 1 inhibitor, and / or PDL-1 inhibitor and / or CTLA-4 inhibitor.

[0022] Still preferably said further therapeutic agent is a chemotherapeutic agent, preferably said chemotherapeutic agent is selected from the group consisting of: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an antibody-drug conjugate (ADC) delivering a topoisomerase inhibitor or a microtubule poison, preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab-paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide, trastuzumab-deruxtecan, sacituzumab-govitecan, datopotamab-deruxtecan, sacituzumab-tirumotecan, enfortumab-vedotin, trastuzumab emtansine (T-DM1).

[0023] Still preferably said further therapeutic agent is a combination of chemotherapeutic agent and an immunotherapy agent, preferably said chemotherapeutic agent is selected from the group consisting of: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an ADC delivering a topoisomerase inhibitor or a microtubule poison, preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab-paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide; and the immunotherapy agent is an immune checkpoint inhibitor (ICI), preferably a PD-1 inhibitor, and / or PDL-1 inhibitor and / or CTLA-4 inhibitor.

[0024] More preferably, said further therapeutic agent is a combination of carboplatin or paclitaxel or nab- paclitaxel or oxaliplatin and a PD-1 inhibitor.

[0025] It is a further object of the invention, leucine or the pharmaceutically acceptable salt thereof for use in the treatment of cancer as defined above, wherein said cancer is selected from the group consisting of: breast cancer, colorectal cancer, small cell and non small cell lung carcinomas (SCLCs and NSCLCs), pancreatic adenocarcinoma, colon cancer, rectal cancer, mucinous adenocarcinoma, melanoma, biliary tract carcinoma, gastric carcinoma, esophageal carcinoma, small intestine carcinoma, urothelial carcinoma, neuroblastomas, gliomas, sarcomas, lymphomas, leukemias, neuroendocrine carcinomas, either early stage or metastatic.

[0026] It is a further object of the invention leucine or the pharmaceutically acceptable salt thereof for use as defined above wherein said cancer is resistant to conventional therapy, preferably said cancer is resistant to radiotherapy or chemotherapy, preferably said cancer is resistant to: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an ADC delivering a topoisomerase inhibitor or a microtubule poison preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab-paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide, trastuzumab-deruxtecan, sacituzumab-govitecan, datopotamab-deruxtecan, sacituzumab-tirumotecan, enfortumab-vedotin, trastuzumab emtansine (T-DM1).

[0027] It is a further object of the invention a pharmaceutical or nutritional composition or dietary supplement comprising leucine or the pharmaceutically acceptable salt thereof for use as defined above, and a pharmaceutical or nutritionally acceptable carrier.

[0028] Preferably the pharmaceutical or nutritional composition or dietary supplement is formulated as an oral formulation with delayed or prolonged intestinal absorption.

[0029] The various embodiments of the invention are presented in the detailed description below and the preferred embodiment are presented in the claims which form an integral part of the present description. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention provides the use of oral leucine supplementation as an umbrella anticancer therapy that could be implemented in several clinical contexts in patients with different tumor types and receiving different standard-of-care concomitant therapies.

[0031] More specifically the invention provides leucine or a pharmaceutically acceptable salt thereof for use in the treatment of cancer, either used alone or in combination with at least one cycle of water- only fasting or fasting mimicking diet (FMD) plus / minus additional therapeutic interventions.

[0032] Preferably said at least one water-only fasting cycle is characterized by 24 h to 72 h, preferably 24 h to 48 h, more preferably 48 h of water-only fasting (100% caloric restriction with unlimited access to water).

[0033] Still preferably said fasting mimicking diet lasts for a period of 24 to 190 hours, preferably said fasting mimicking diet lasts for a period of 24 to 150 hours, preferably said fasting mimicking diet lasts for approximately 120 hours (5 days).

[0034] In a further preferred embodiment of the invention said fasting mimicking diet is a regular caloric intake reduced by 65% to 85% and / or said fasting mimicking diet is based on an average caloric intake of between 300-600 Kcal / day, wherein said caloric intake is preferably referred to humans. Preferably the fasting mimicking diet comprises a first period of 0 to 24 hours wherein the caloric intake is a regular caloric intake reduced by approximately 65%, followed by a second period of 24 to 96 hours wherein the caloric intake is a regular caloric intake reduced by approximately 85%, preferably the first period lasts approximately 24 hours and the second period lasts approximately between 48 and 96 hours.

[0035] In a further preferred embodiment of the invention said fasting mimicking diet comprises a reduced protein intake and / or a reduced simple carbohydrate (sugars) intake, and / or a relatively increased content of unsaturated fat intake.

[0036] Still preferably said fasting mimicking diet comprises administering a food having a relative content of monounsaturated and / or polyunsaturated fats between 10 and 80 %, a content of proteins from 0 to 10 % and a content of carbohydrates from 5 to 50 %.

[0037] It is a further object of the invention leucine or the pharmaceutically acceptable salt thereof for use as defined above wherein leucine is administered in an amount of approximately between 25 mg / kg and 800 mg / kg on a daily basis, preferably between 50 mg / kg and 500 mg / kg on a daily basis.

[0038] It is a further object of the invention or the pharmaceutically acceptable salt thereof for use as defined above wherein leucine is administered in an amount of approximately between 10 mg / kg and 1000 mg / kg on a daily basis, preferably between 25 mg / kg and 800 mg / kg on a daily basis, preferably between 50 mg / kg and 500 mg / kg on a daily basis, more preferably between 50 and 100 mg / kg.

[0039] It is a further object of the invention leucine or the pharmaceutically acceptable salt thereof for use as defined above and / or in combination with a further therapeutic intervention selected from the group consisting of: surgery, radiotherapy and a further therapeutic agent.

[0040] Preferably said further therapeutic agent is an immune checkpoint inhibitor (ICI), preferably a PD- 1 inhibitor, and / or PDL-1 inhibitor and / or CTLA-4 inhibitor.

[0041] Still preferably said further therapeutic agent is a chemotherapeutic agent, preferably said chemotherapeutic agent is selected from the group consisting of: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an antibody-drug conjugate (ADC) delivering a topoisomerase inhibitor or a microtubule poison, preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab-paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide, trastuzumab-deruxtecan, sacituzumab-govitecan, datopotamab-deruxtecan, sacituzumab-tirumotecan, enfortumab-vedotin, trastuzumab emtansine (T-DM1).

[0042] Still preferably said further therapeutic agent is a combination of chemotherapeutic agent and an immunotherapy agent, preferably said chemotherapeutic agent is selected from the group consisting of: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an ADC delivering a topoisomerase inhibitor or a microtubule poison, preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab-paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide; and the immunotherapy agent is an immune checkpoint inhibitor (ICI), preferably a PD-1 inhibitor, and / or PDL-1 inhibitor and / or CTLA-4 inhibitor.

[0043] More preferably, said further therapeutic agent is a combination of carboplatin or paclitaxel or nab- paclitaxel or oxaliplatin and a PD-1 inhibitor.

[0044] It is a further object of the invention, leucine or the pharmaceutically acceptable salt thereof for use in the treatment of cancer as defined above, wherein said cancer is selected from the group consisting of: breast cancer, colorectal cancer, small cell and non small cell lung carcinomas (SCLCs and NSCLCs), pancreatic adenocarcinoma, colon cancer, rectal cancer, mucinous adenocarcinoma, melanoma, biliary tract carcinoma, gastric carcinoma, esophageal carcinoma, small intestine carcinoma, urothelial carcinoma, neuroblastomas, gliomas, sarcomas, lymphomas, leukemias, neuroendocrine carcinomas, either early stage or metastatic. It is a further object of the invention leucine or the pharmaceutically acceptable salt thereof for use as defined above wherein said cancer is resistant to conventional therapy, preferably said cancer is resistant to radiotherapy or chemotherapy, preferably said cancer is resistant to: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an ADC delivering a topoisomerase inhibitor or a microtubule poison preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab-paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide, trastuzumab-deruxtecan, sacituzumab-govitecan, datopotamab-deruxtecan, sacituzumab-tirumotecan, enfortumab-vedotin, trastuzumab emtansine (T-DM1).

[0045] Preferably said cancer is selected from the group consisting of: breast cancer, colorectal cancer, small cell and non small cell lung carcinomas (SCLCs and NSCLCs), pancreatic adenocarcinoma, colon cancer, rectal cancer, mucinous adenocarcinoma, melanoma, biliary tract carcinoma, gastric carcinoma, esophageal carcinoma, small intestine carcinoma, urothelial carcinoma, neuroblastomas, gliomas, sarcomas, lymphomas, leukemias, neuroendocrine carcinomas, preferably said cancer is either primary and metastatic cancer.

[0046] It is a further object of the invention a pharmaceutical or nutritional composition or dietary supplement comprising leucine or the pharmaceutically acceptable salt thereof for use as defined above, and a pharmaceutical or nutritionally acceptable carrier.

[0047] Preferably the pharmaceutical or nutritional composition or dietary supplement is formulated as an oral formulation with delayed or prolonged intestinal absorption.

[0048] The present invention provides leucine and derivatives thereof or a pharmaceutically acceptable salt of leucine or its derivatives, for use as a food supplement in a method for treating cancer. Leucine may be in racemic form, which means that the compound comprises about equal amounts of enantiomers. Alternatively it may be present in an enantiomeric excess of either the L- enantiomer or the D-enantiomer. In one embodiment, leucine is present in an enantiomeric excess of the L-enantiomer. The racemic and enantiomeric forms may be obtained in accordance with known procedures in the art. Leucine derivatives include carboxylgroup derivatives and aminogroup derivatives. Carboxyl group derivatives include L-leucine methyl ester (H-Leu-OMe), L- leucine ethyl ester (H-Leu-OEt), L-leucine allyl ester (H-Leu-OAll), L-leucine 2-propoxyethyl ester (H-Leu-O(CH2)2OPr), L-leucine t-butyl ester (H-Leu-OtBu), L-leucine benzyl ester (H-Leu- OBzl), 1-leucinylmethoxymethane (H-Leu-OCEbOMe), L-leucine hydroxamate (H-Leu-NHOH). Amino group derivatives include N-acetyl-L-leucine (Ac-Leu-OH), N-formyl-L-leucine (For-Leu- OH), N-Methyl-L-leucine (Me-Leu-OH). A "pharmaceutically acceptable salt" as referred to herein, is any salt preparation that is appropriate for use in a pharmaceutical application or in a food supplement. Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzylphenethylamine, 1-para- chloro- benzyl-2-pyrrolidin-i'- ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine, tri s(hydroxymethyl)aminom ethane and the like; alkali metal salts, such as lithium, potassium, sodium and the like; alkali earth metal salts, such as barium, calcium, magnesium and the like; transition metal salts, such as zinc, aluminum and the like; other metal salts, such as sodium hydrogen phosphate, disodium phosphate and the like; mineral acids, such as hydrochlorides, sulfates and the like; and salts of organic acids, such as acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates, fumarates and the like.

[0049] A "pharmaceutically or nutritionally acceptable carrier" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions or nutritional compositions such as food supplements. In a further embodiment, the pharmaceutically or nutritionally acceptable carrier may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically or nutritionally acceptable carrier may include, but is not limited to, one or more substances which may also act as flavouring agents, buffers, lubricants, stabilisers, solubilisers, suspending agents, wetting agents, emulsifiers, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The carrier may also be an encapsulating material. In powders, the carrier is a finely divided solid that is in admixture with the finely divided active agents according to the present disclosure. In tablets, the active agent may be mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets, for example, contain up to 99% of the active agents. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutically or nutritionally acceptable carrier maybe a gel and the composition maybe in the form of a cream or the like. In yet a further embodiment, the carrier may include, but is not limited to, one or more excipients or diluents. Examples of such excipients are gelatin, gum arabicum, lactose, microcrystalline cellulose, starch, sodium starch glycolate, calcium hydrogen phosphate, magnesium stearate, talcum, colloidal silicon dioxide and the like. The pharmaceutical or nutritional composition or dietary supplement can also be formulated as an oral formulation with delayed, prolonged or optimized intestinal absorption. As formulation with delayed or prolonged intestinal absorption it is intended that the composition is formulated in a way allowing a release into the body of a specified amount over a specified period of time of an active ingredient, namely a specific pharmacokinetic profile. The above expression is equivalent to controlled release and encompasses all the type of releases that are modified in comparison to an immediate release.

[0050] This type of release means that the release is prolonged over time in comparison to an immediate release, i.e. it means that the active ingredient is released slowly over time, allowing a less frequent intake of the composition for the patient, or simply an optimized absorption of leucine to achieve more durable therapeutic concentrations of leucine in the blood and in the tumor microenvironment.

[0051] In another embodiment, the pharmaceutically or nutritionally acceptable carrier may be a liquid, and the pharmaceutical composition or the food supplement is in the form of a solution. Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the present disclosure may be dissolved or suspended in a liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurised compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0052] Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be administered through, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and subcutaneous injection. The active agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. The agents and compositions of the present disclosure may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the disclosure can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0053] Leucine, including its salts and derivatives, and compositions comprising the same may alternatively be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.

[0054] In one embodiment, the pharmaceutical composition or the nutritional composition is in the form of a tablet. In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The tablets may contain up to 99% by weight of leucine or a salt thereof or a derivative thereof.

[0055] Thus, in one embodiment, leucine, or the pharmaceutically acceptable salt of leucine, is provided in a solid dosage form suitable for oral administration, notably in the form of a tablet.

[0056] The above-mentioned pharmaceutical or nutritional formulations in solid oral dosage form, such as tablets, may be prepared by any method known in the art of pharmacy. Said formulations are usually prepared by mixing the active substance, or a pharmaceutically acceptable salt thereof, with conventional pharmaceutically acceptable carriers, diluents or excipients.

[0057] Leucine, or a pharmaceutically acceptable salt of the same, may be administered at a daily dose between 10 mg / kg and 1000 mg / kg, preferably between 25 mg / kg and 800 mg / kg, preferably between 50 mg / kg and 500 mg / kg.

[0058] Said dosages may be administered by solid oral or liquid oral route.

[0059] The total daily dose may be divided in multiple administrations, e.g. one administration may be required two or more times a day to achieve the required dose. As an example, the required number of tablets to provide the total daily dose of leucine may be split into two daily administrations (for example, in the morning and evening) or three administrations (for example, in the morning, noon and evening).

[0060] Treatment duration may vary according to tumor response, duration of tumor response, relapse- free survival (RFS), disease-free survival (DFS) and Event-free survival (EFS) in patients with surgically resected tumor, progression-free survival (PFS) and overall survival (OS) in patients with advanced malignancies, or other clinical factors, including treatment tolerability and quality of life. It may be seven days or more, two weeks or more, three weeks or more, one month or more, six weeks or more, seven weeks or more or two months or more. For example, it is three months or more, four months or more, five months or more or even six months or more.

[0061] Any and all combinations of dosage form, dose amount, dosing schedule and treatment duration are envisaged and encompassed by the disclosure. An example combination is a total daily dose between 3.5 g and 35 g per day, preferably 4.0 g and 20 g per day, preferably between 4 g and 18 g, preferably between 4 g and 15 g, preferably between 4.5 g and 10 g per day, taken across three administrations per day, for a treatment duration of two months or more. A further example combination is a total daily dose of more than 4 g to no more than 35 g per day, taken across three administrations per day, for a treatment duration of six months or more. The dosage form may be, for example, a solid oral dosage form, notably tablets.

[0062] A "subject", as used herein, may be a vertebrate, mammal or domestic animal. Hence, compositions according to the disclosure may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Further for example, the subject is a human. In the present invention leucine is used as food supplement for treatment of cancer in combination with a specific caloric intake regime. Said specific caloric intake is based on a reduced daily caloric intake compared to a regular daily caloric intake, in particular it involves a specific daily caloric intake and a specific macronutrient intake as defined below.

[0063] In the present invention, a specific caloric and macronutrient intake may be achieved, for example, by means of cyclic water-only fasting or of a fasting mimicking diet (FMD), which are equivalent in terms of biological and antitumor effects.2’6’26

[0064] Fasting, or a fasting cycle according to the invention, involves about 2 days of nutrient starvation, with free consumption to water. Therefore, said fasting cycle is 100% reduced caloric intake.

[0065] “Fasting mimicking diet” (FMD) refers to previously described formulations to mimic the effects of water-only fasting.8Water-only fasting results to be challenging for cancer patiens, especially when undergoing chemotherapy, so the inventors have developed a FMD that enables a patient to eat “food” while achieving the same effects of fasting on normal and cancer cells.

[0066] Water-only fasting or FMD are initiated one or two days before the administration of pharmacological therapies (e.g., chemotherapy, immunotherapy, chemoimmunotherapy, taegeted therapies) and continues for the following 2-7 days, preferably for the following 2-4 days while the therapy is most active. In a preferred embodiment, FMD comprises one or more FMD cycles, each cycle consisting of 1- 7 days (preferably of 2-5 days, more preferably 5 days) of low-calorie intake, for example as follows:

[0067] Day 1 :

[0068] Mouse = approximately 50% of regular caloric intake

[0069] Human = approximately 35% of regular caloric intake (i.e. regular caloric intake reduced by approximately 65%)

[0070] Days 2-5:

[0071] Mouse = approximately 10% of regular caloric intake (i.e. regular caloric intake reduced by approximately 90%)

[0072] Human = approximately 15% of regular caloric intake (i.e. regular caloric intake reduced by approximately 85%)

[0073] A further example of FMD cycles in human is as follows:

[0074] Day 1 :

[0075] Human = approximately 50% of regular caloric intake

[0076] Days 2-5:

[0077] Human = approximately 30% of regular caloric intake (i.e. regular calorie intake reduced by approximately 65%)

[0078] Other known protocols of FMD can be used within the invention. For example a further preferred reduced caloric intake is as follows:

[0079] Day 1 : 54% caloric intake, about 1,090 kcal (10% protein, 56% fat, 34% carbohydrate)

[0080] Days 2-7: 20-34% caloric intake, about 426-725 kcal (5.3-9% protein, 26-44% fat, 27.6-47% carbohydrate).

[0081] In the present invention preferably the reduced caloric intake is obtained by water-only fasting or by means of dietetic food with reduced caloric and / or protein content and / or carbohydrate content but containing all necessary micro nutrients to prevent malnutrition.

[0082] FMD is achieved with a low protein and low sugar and relatively high fat plant-based formulation followed by a standard / regular ad libitum diet until the complete recovery of body weight.

[0083] The reduction is compared to a regular caloric intake per day. A regular caloric intake for an adult, calculated on average for male and female, is of about 1800 Kcal / day. As used herein, regular intake is intended as about 1800 Kcal / day.

[0084] In the present invention the period of reduced caloric intake is repeated one or more times after respective periods of 3-60 days of regular caloric intake. According to the invention, Leucine supplementation is given to the subject both during the period(s) of reduced caloric intake and during the period(s) of regular caloric intake.

[0085] In a preferred embodiment said mammal is fed with a food having a relative content of monounsaturated and / or polyunsaturated fats between 10 and 80 %, a content of proteins from 0 to 10 % and a content of carbohydrates from 5 to 50%.

[0086] Examples of useful fasting mimicking diets in the context of the present invention are set forth in U. S. Pat. Appl. Nos. 14 / 273,946 filed May 9, 2014; 14 / 497,752 filed September 26, 2014; 12 / 910,508 filed October 22, 2010; 13 / 643,673 filed October 26, 2012; 13 / 982,307 filed July 29, 2013; 14 / 060,494 filed October 22, 2013; 14 / 178,953 filed February 12, 2014; 14 / 320,996 filed July 1, 2014; 14 / 671,622 filed March 27, 2015. Additionally informative examples of FMD diets are also found in US patent application Ser. No. 15 / 148,251 and WIPO Pub. No. WO2011 / 050302.; W02020 / 261131.

[0087] Usually, humans undergoing one or more FMD cycles do not lose more than 10% of body weight. FMD cycles are feasible and safe thus individuals well tolerate the diet. Reasons for stopping the FMD are not related to heathy status but usually to non-compliance to the dietary protocol or for work scheduling issues. However, there are clinical condition which can make the individual not eligible to FMD, such as being underweight, or being unable to at least partially restore the weight that was lost during the FMD.

[0088] The present invention will be illustrated by means of non-limiting examples and figures as follows.

[0089] FIG 1. Leucine, alone or combined with 48h water-only fasting, delays cancer progression and significantly prolongs mouse survival in 4 different syngeneic models.

[0090] A) Growth of 4Tl-luc (constitutively expressing luciferase) cell transplants in the mammary fat pad of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), alone or combined with Leucine supplementation (500 mg / kg) administered once a day, as given through oral gavage (n=14). B) Survival curves of mice treated as described in A) are reported. C) Growth of E0771 cell transplants after cell injection in the mammary fat pad of 7-weeks old female C57BL6 / J mice fed with standard diet or fasting cycles, alone or combined with oral Leucine (500 mg / kg) administered once a day (n=9-10). D) Survival curves of mice treated as described in C) are reported. E) Growth of CT26 CRC cell transplants after cell injection in the right flank of 7- weeks old female BALB / c mice fed with standard diet or fasting cycles, alone or combined with Leucine (500 mg / kg) administered once a day, by oral gavage (n=6). F) Survival curves of mice treated as described in E) are reported. G) Growth of LLC lung cell transplants after cell injection in the right flank (subcutaneous tissue) of 7-weeks old female C57BL6 / J mice fed with standard diet or fasting cycles, alone or combined with leucine (500mg / kg) administered once a day, by oral gavage (n=5). H) Survival curves of mice treated as described in G) are reported.

[0091] Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova. Comparison of survival curves were performed with Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant. I) Growth of 4Tl-luc (constitutively expressing luciferase) cell transplants in the mammary fat pad of 7-weeks old female BALB / c mice fed with standard diet alone or combined with Leucine supplementation (500 mg / kg) administered once a day, as given through oral gavage or chronic Leucine supplementation given in mice drinking water at 2.5 mg / ml (n=14). J) Survival curves of mice treated as described in I) are reported.

[0092] FIG 2. Impact of Leucine supplementation, alone or combined with anti-PD-Ll immunotherapy, on tumor growth and animal survival.

[0093] A) Growth of 4Tl-luc cell transplants after cell injection in the mammary fat pad of 7-weeks old female BALB / c mice treated with oral Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles, (n=18-23). B) Survival curves of animals, as treated as described in A), are reported. C) Growth of E0771 cell transplants in the mammary fat pad of 7-weeks old female C57BL6 / J mice treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus anti-PDLl MoAb (lOOug per mouse) every other day, for a total of 4 cycles, by intraperitoneal injection (n=9-10). D) Survival curves of mice treated as described in C) are reported. E) Growth of CT26 CRC cell transplants in the right flank of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles, alone or combined with Leucine (500 mg / kg), as administered once per day by oral gavage, plus / minus anti-PDLl MoAb (lOOug per mouse) every other day, for a total of 4 cycles, by intraperitoneal injection (n=6). F) Survival curves of animals treated as described in E) are reported.

[0094] G) Growth of LLC lung cell transplants after cell injection in the right flank (subcutaneous tissue) of 7-weeks old female C57BL6 / J fed with standard diet or fasting cycles, alone or combined with Leucine (500 mg / kg), as administered once per day by oral gavage, plus / minus anti-PDLl MoAb (lOOug per mouse) every other day, for a total of 4 cycles, by intraperitoneal injection (n=10-15).

[0095] H) Survival curves of mice treated as described in G) are reported.

[0096] Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova. Comparison of survival curves were performed with Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant.

[0097] FIG 3. Impact of Leucine supplementation, alone or combined with platinum chemotherapy, on tumor growth and animal survival.

[0098] A) Growth of 4Tl-luc cell transplants in the mammary fat pad of 7-weeks old female BALB / c mice treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus carboplatin (50 mg / kg), as administered once a week by intraperitoneal injection (n=6). B) Survival curves of animals treated as described in A) are reported. C) Growth of E0771 cell transplants in the mammary fat pad of 7-weeks old female C57BL6 / J mice treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus paclitaxel (30 mg / kg), as administered once a week by intraperitoneal injection (n=9-12). D) Survival curves of animals treated as described in C) are reported. E) Growth of CT26 cell transplants in the right flank of 7-weeks old female BALB / c mice treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus oxaliplatin (5 mg / kg), as administered once a week by intraperitoneal injection (n=6). F) Survival curves of animals treated as described in E) are reported. G) Growth of LLC lung cell transplants after cell injection in the right flank (subcutaneous tissue) of 7-weeks old female C57BL6 / J treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus carboplatin (50 mg / kg), as administered once a week by intraperitoneal injection (n=10- 15). H) Survival curves of mice treated as described in G) are reported.

[0099] Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova. Comparison of survival curves were performed with Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant.

[0100] FIG 4. Impact of Leucine supplementation, alone or combined with platinum chemotherapy and anti-PD-Ll immunotherapy, on tumor growth and animal survival.

[0101] A) Growth of 4Tl-luc cell transplants in the mammary fat pad of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus carboplatin (50 mg / kg), as administered once a week by intraperitoneal injection, plus / minus murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles (n=6-8). B) Survival curves of animals treated as described in A) are reported. C) Growth of E0771 cell transplants in the mammary fat pad of 7-weeks old female C57BL6 / J mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus paclitaxel (30 mg / kg), as administered once a week by intraperitoneal injection, plus / minus murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles (n=9-12). D) Survival curves of animals treated as described in C) are reported. E) Growth of CT26 cell transplants in the right flank of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus oxaliplatin (5 mg / kg), as administered once a week by intraperitoneal injection, plus / minus murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other- day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles (n=6-7). F) Survival curves of animals treated as described in E) are reported. G) Growth of LLC lung cell transplants after cell injection in the right flank (subcutaneous tissue) of 7-weeks old female C57BL6 / J fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), treated with Leucine (500 mg / kg), as administered once a day by oral gavage, plus / minus carboplatin (50 mg / kg), as administered once a week by intraperitoneal injection, plus / minus murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles (n=10-15). H) Survival curves of mice treated as described in G) are reported.

[0102] Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova or unpaired t-test for comparisons between only 2 groups.

[0103] Comparison of survival curves were performed with Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant.

[0104] FIG 5. Fasting plus Leu supplementation enhances the anti-cancer effect of chemoimmunotherapy (ChT-IO).

[0105] A) Growth of 4Tl-luc cell transplants in the mammary fat pad of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), combined with Leu supplementation (500 mg / kg), as administered once a day by oral gavage, plus / minus carboplatin (50 mg / kg), as administered once a week by intraperitoneal injection, in combination with murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles (n=6-8). B) Survival curves of animals treated as described in A) are reported. C) Growth of E0771 cell transplants in the mammary fat pad of 7-weeks old female C57BL6 / J mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), combined with Leu (500 mg / kg), as administered once a day by oral gavage, plus / minus paclitaxel (30 mg / kg), as administered once a week by intraperitoneal injection, in combination with murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles (n=9-12). D) Survival curves of animals treated as described in C) are reported. E) Growth of CT26 cell transplants in the right flank of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), combined with Leu (500 mg / kg), as administered once a day by oral gavage, plus / minus oxaliplatin (5 mg / kg), as administered once a week by intraperitoneal injection, in combination with murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other day by intraperitoneal injection, for a total number of anti-PD-Ll treatment cycles (n=6-7). F) Survival curves of animals treated as described in E) are reported. G) Growth of LLC lung cell transplants after cell injection in the right flank (subcutaneous tissue) of 7-weeks old female C57BL6 / J fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), combined with Leu (500 mg / kg), as administered once a day by oral gavage, plus / minus carboplatin (50 mg / kg), as administered once a week by intraperitoneal injection, in combination with murine anti-PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for a total number of 4 anti-PD-Ll treatment cycles (n=10-15). H) Survival curves of mice treated as described in G) are reported. I) Growth of 4Tl-luc cell transplants in the mammary fat pad of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), combined with Leu supplementation (250 / 500 / 700 mg / kg), as administered once a day by oral gavage, plus / minus carboplatin (50 mg / kg), as administered once a week by intraperitoneal injection, in combination with murine anti- PDLl Monoclonal Antibody (MoAb) (lOOug in each mouse) every-other-day by intraperitoneal injection, for atotal number of 4 anti-PD-Ll treatment cycles (n=6-8). J) Survival curves of animals treated as described in I) are reported. Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova or unpaired t-test for comparisons between only 2 groups. K) Plasmatic glucose levels measured in mice treated as described in I). L) Plasmatic insulin glucose levels measured in mice treated as described in I).

[0106] Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova. Comparison of survival curves were performed with Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant.

[0107] FIG 6. The anticancer effects of Leu supplementation are mediated by CD8+ T lymphocytes.

[0108] A) Growth of 4Tl-luc (constitutively expressing luciferase) cell transplants in the mammary fat pad of 7-10 weeks old female NSG mice fed with standard diet or fasting cycles (48-hours of water- only fasting, followed by ad libitum refeeding, and repeated every 7 days), alone or combined with Leucine supplementation (500 mg / kg) administered once a day, as given through oral gavage (n=9).

[0109] B) Survival curves of mice treated as described in A) are reported. C) Growth of E0771 cell transplants after cell injection in the mammary fat pad of 7-10 weeks old female NSG mice fed with standard diet alone or combined with oral Leucine (500 mg / kg) administered once a day (n=9- 10). D) Survival curves of mice treated as described in C) are reported. E) Growth of 4Tl-luc (constitutively expressing luciferase) cell transplants in the mammary fat pad of 7 weeks old female BALB / c mice treated with non-specific murine IgG. Mice were fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), alone or combined with Leucine (500 mg / kg) administered once a day, by oral gavage (n=9- 10). F) Survival curves of mice treated as described in E) are reported. G) Growth of 4Tl-luc (constitutively expressing luciferase) cell transplants in the mammary fat pad of 7 weeks old female BALB / c mice in which CD8+ T cells were depleted through an anti-CD8 murine monoclonal antibody (MoAb). Mice were fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), alone or combined with leucine (500 mg / kg) administered once a day, by oral gavage (n=10). H) Survival curves of mice treated as described in G) are reported. I) Adoptive cell transfer of untreated CD8 T cells isolated from the spleens and lymph nodes of immunocompetent C57BL6 / j mice. CD8 T cells were injected on days 5 and day 10 in NSG mice previously orthotopically injected with 4Tl-luc cells; starting from day 5, NSG mice were fed with standard diet alone or combined with Leucine supplementation (500 mg / kg) administered once a day, + / - fasting cycles. CTRL no transfer indicates tumor growth in NSG mice without T cell administration (n=9-10). J) Survival curves of mice treated as described in I) are reported.

[0110] Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova. Comparison of survival curves were performed with Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant.

[0111] FIG 7. Leu supplementation primes tumor cells to be recognized by CD8+ T lymphocytes. A) Adoptive cell transfer of CD8 T cells isolated from the spleens and lymph nodes of immunocompetent C57BL6 / j mice previously exposed to CTRL, Leu supplementation, or Leu + fasting combination. CD8 T cells were infused on days 5 and day 10 in NSG mice previously orthotopically injected with 4Tl-luc cells, and never exposed to Leu supplementation. CTRL: no transfer indicates tumor growth in NSG mice without T cell administration (n=10). B) Survival curves of mice treated as described in A) are reported. C) Adoptive cell transfer of untreated CD8 T cells isolated from the spleens and lymph nodes of immunocompetent C57BL6 / j mice. CD8 T cells were injected on days 7 and 12 into E0771-bearing NSG mice that were exposed to Leu or fasting + Leu only until CD8+ T cells transfer. D) Survival curves of mice treated as described in C) are reported. E) Growth of primary mammary tumors resulting from the injection of 4T1 cells engineered with an empty plasmid (blank), after cell injection in the mammary fat pad of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), alone or combined with Leucine supplementation (500 mg / kg) administered once a day, as given through oral gavage (n=10). F) Survival curves of mice treated as described in E) are reported. G) Growth of primary mammary tumors resulting from the injection of 4T1 cells engineered to overexpress BCAT1 (BCAT1-OE), after cell injection in the mammary fat pad of 7-weeks old female BALB / c mice fed with standard diet or fasting cycles (48-hours of water-only fasting, followed by ad libitum refeeding, and repeated every 7 days), alone or combined with Leucine supplementation (500 mg / kg) administered once a day, as given through oral gavage (n=10). H) Survival curves of mice treated as described in G) are reported. I) Adoptive cell transfer of CD8 T cells isolated from the spleens and lymph nodes of immunocompetent E0771 -bearing C57BL6 / j mice exposed to CTRL, Leu supplementation, or Leu + fasting combination. CD8 T cells were infused at day 5 and day 10 in NSG mice previously orthotopically injected with 4Tl-luc cells, never exposed to Leu supplementation. CTRL no transfer indicates tumor growth in NSG mice without T cell administration (n=10). J) Survival curves of mice treated as described in I) are reported.

[0112] Data are represented as mean ± SEM. P values were determined by ordinary one-way Anova. Comparison of survival curves were performed with Log-rank (Mantel-Cox) test. P values < 0.05 were considered significant.

[0113] EXAMPLES

[0114] Mouse models Animals were housed under specific pathogen-free conditions with 12 hours day / light cycles. All experiments were performed in accordance with the Italian Laws (D.lgs. 26 / 2014), which enforce Directive 2010 / 63 / EU (Directive 2010 / 63 / EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes) and they were approved by the Italian Ministry of Health.

[0115] As for the syngeneic 4T1 TNBC model, 7-weeks old female BALB / c / Ola Hsd mice (Envigo) were injected in the mammary fat pad with 2xlO44Tl-luc (luciferase-positive) cells resuspended in 20 pl of medium (RPMI supplemented with 10% FBS, 2mM glutamine). As for the immunodeficient 4T1 TNBC model, 7-10-weeks old female NSG mice were injected in the mammary fat pad with 2X1044T1-1UC cells resuspended in 20 pl of medium. As for the experiment employing BCAT1 overexpressing cells, 4T1 parental cells and 4T1 cells engineered with an empty plasmid (blank; Abm good # LV587) or to overexpress BCAT1 (BCAT1-OE; Abm good #132140640195) were injected in the mammary fat pad of female BALB / c mice. As for syngeneic E0771 TNBC model, 7-weeks old female C57BL6 / J (Charles river) were injected in the mammary fat pad with 3xl05E0771 cells resuspended in 40 pl of medium (RPMI supplemented with 10% FBS, 2mM glutamine). As for the immunodeficient E0771 TNBC model, 7-10-weeks old female NSG mice were injected in the mammary fat pad with 3xl05E0771 cells resuspended in 40 pl of medium. As for the syngeneic lung cancer model, 7-weeks old female C57BL6 / J (Charles River) were injected subcutaneously with 2xlO5LLC cells resuspended in 100 pl of PBS. Finally, for the establishment of the syngeneic colorectal cancer model, 7-weeks old female BALB / c / Ola Hsd mice (Envigo) were injected subcutaneously with 3xl05CT26 cells resuspended in 100 pl of PBS. When tumors were palpable (7 days after cells inoculation for 4T1, E0771 and CT26 models, 5 days after cells inoculation for LLC model), mice were randomly assigned to different experimental groups. Tumor volumes were measured twice a week through a digital caliper according to the following equation: tumor volume (mm3) = length x width x thickness x 0,5. At the end of the experiments, mice were euthanized by using CO2.

[0116] Animal diets and treatments

[0117] Mice were fed ad libitum with irradiated standard diet VRFI (P) diet (Charles River) containing 3,89 kcal / g of gross energy. According to the experimental groups, some mice were subjected to fasting one per week, consisting of 48-hour water-only-fasting, followed by 5 days of ad libitum refeeding (standard diet), and repeated until unacceptable animal toxicity or sacrifice. Mouse weight was monitored twice per week and during any fasting cycle. In these experiments, weight loss could not exceed 20% as compared to baseline values. Before initiating a new fasting cycle, the animals should have completely recovered their original bodyweight.

[0118] As for experiments employing the use of Leucine, mice were treated on a daily basis with 500 mg / kg of Leucine (Sigma Aldrich, # L8000), which was dissolved in physiologic water and administered by oral gavage. As for experiments involving Leu supplementation in mice drinking water, it was dissolved at 2.5 mg / ml corresponding to a daily dose of 500 mg / kg (lOmg / mouse) considering that mice normally drink 4 ml / day of water. In the experiment described in Fig 5 L, Leu was administered at different doses, 250, 500 and 700 mg / kg which was dissolved in physiologic water and administered by oral gavage.

[0119] As for the experiments employing anti-PDLl immunotherapy, mice were treated with InVivoMAb anti-mouse PD-L1 (B7-H1) (BioXCell, # BE0101), at a dose of 100 ug per mouse, via intraperitoneal injection. Mice were treated with anti-PDLl every other day, up to a maximum of 4 cycles (day 0, 2, 4, 6 after the beginning of the experiments).

[0120] As for experiments employing the use of chemotherapy, carboplatin (Sindan, 10 mg / ml), oxaliplatin (Mylan, 5 mg / ml in infusion solution) and paclitaxel (Accord, 6 mg / ml in 527 mg / ml poly oxy ethylated castor oil and 391 mg / ml ethanol) were provided by Istituto Nazionale dei Tumori, Milano.

[0121] Carboplatin was administered at a dose of 50 mg / kg (final volume lOOul), once x week, via intraperitoneal injection; oxaliplatin was administered at a dose of 5 mg / kg (diluted in injectable solution, final volume lOOul), once per week, via intraperitoneal injection; paclitaxel was administered at a dose of 30 mg / kg (diluted in physiologic water, final volume 200ul), once x week, via intraperitoneal injection.

[0122] For the experiment employing the use of anti-CD8 antibody and IgG, as control, mice were treated with InVivoMAb anti-mouse CD8a (BioXCell #BE0117) and / wEzvoMAb rat IgG2b (BioXCell #BE009). Depletion of CD8+ T cells was achieved by intraperitoneal injection of anti-CD8 at a concentration of 200ug per mouse, 3 days before tumor cells inoculation and every 3 days thereafter (for a total of 5 cycles).

[0123] Adoptive T cell transfer

[0124] Donor C57BL6 / j mice were euthanized and their spleen and lymph nodes were collected to isolate CD8 T cells. Briefly, spleens and lymph nodes were minced and passed through a 70-pm cell strainer. Red blood cells were lysed with ACK lysis buffer. T cells isolation was performed using negative selection with magnetic beads. CD8a+T Cell Isolation Kit mouse was used (Miltenyi # 130- 104-075). 5xl06cells were dissolved in 200 pl of PBS and intravenously injected in recipient E0771 -bearing NSG mice. CD8 T cells adoptive transfer was performed twice, on days 5 and 10, upon cancer cells injection in recipient NSG mice in experiments reported at Fig 6 I-J, Fig 7 A-B and Fig 7 I-J, while it was performed on days 7 and 12 in experiments reported at Fig 7 C-D. In Fig 6 I-J, healthy donor C57BL6 / j mice never exposed to Leu were used, while recipient E0771- bearing NSG mice were treated with Leu supplementation alone or in combination with fasting starting from day 5 after cells inoculation. In Fig 7 A-B, healthy donor C57BL6 / j mice exposed to Leu or fasting + Leu for a total of 5 supplementation (48h fasting cycle + 2 days refeeding) were used while recipient E0771-bearing NSG mice were never exposed to Leu. In Fig 7 C-D, healthy donor C57BL6 / j mice never exposed to Leu, while recipient E0771-bearing NSG mice were treated with Leu supplementation alone or in combination with 48h fasting for a total of 5 consecutive days, before CD8 T cells transfer. In Fig 7 I- J, E0771 -bearing donor C57BL6 / j mice exposed to Leu or fasting + Leu for a total of 12 supplementation (48h fasting + 5 days refeeding + 48h fasting cycle + 2 days refeeding) were used while recipient E0771-bearing NSG mice were never exposed to Leu.

[0125] Results

[0126] Leucine supplementation retards tumor growth and synergizes with cyclic fasting in syngeneic TNBC, CRC and lung cancer models

[0127] The impact of oral Leucine supplementation, as given at a daily dose of 500 mg / Kg Leucine by oral gavage, was investigated on in vivo tumor growth and mouse survival in orthotopic (4T1 and E0771 TNBC cells injected in the mammary fat pad of female BALB / c mice and female C57BL6 / J mice, respectively) or subcutaneous (CT26: murine colorectal carcinoma - CRC, or LLC: murine squamous lung carcinoma cells, injected in the subcutaneous tissue of female BALB / c mice and female C57BL6 / J mice, respectively) syngeneic tumor models.

[0128] As shown in Figure 1, Leucine supplementation slowed down primary tumor growth and prolonged animal survival in all these models. As previously reported by other groups3, 4’7, cyclic fasting, consisting of 48-hour water-only fasting followed by ad libitum feeding, also slowed down tumor growth and prolonged animal survival. Of note, combining Leucine supplementation and cyclic fasting resulted in cooperative reduction of in vivo tumor growth, as well as in significant prolongation of animal survival (Figure 1).

[0129] Considering that Leu supplementation could be used in the clinic as a potential anti-cancer intervention against many types of cancer, different schedules of Leu administration were studied to confirm its anti-cancer effects even if used in a different modality. Since Leu given orally every day at such a high concentration could be poorly feasible in patients, a chronic Leu administration potentially capable of replicating the antitumor activity of daily Leu supplementation given by oral gavage was tested. In particular, Leu was added in drinking water and chronically administered for consecutive 48h, every week, dissolved at 2.5mg / ml corresponding to a dose of 500mg / kg (lOmg / mouse) considering that mice normally drink 4ml / day of water. Chronic Leu supplementation slowed down primary tumor growth and prolonged animal survival in 4T1 TNBC model, similarly to daily Leu given orally by acute gavage (Fig 1 I- J).

[0130] Together, these results indicate that oral Leucine supplementation can enhance the antitumor activity of cyclic fasting / FMD, which has recently emerged as a promising antitumor strategy in several clinical settings6, 8’9’20’21.

[0131] Leucine supplementation cooperates with anti-PD-Ll immunotherapy to retard tumor growth and to prolong animal survival in syngeneic mouse tumor models

[0132] Since immune checkpoint inhibitors, such as anti-PDl or anti-PD-Ll Monoclonal Antibodies (MoAb), have recently emerged as effective antitumor treatments in patients with different malignancies, including early stage / advanced TNBC12'14, lung carcinomas15'18and CRC19, the inventors investigated whether Leucine supplementation enhances the antitumor effects of anti- PD-Ll immunotherapy. For these experiments, syngeneic 4T1 (BALB / c mice), E0771 (C57BL / 6 mice), CT26 (BALB / c) and LLC (C57BL / 6 mice) cell transplants were used. As shown in Figure 2, anti-PD-Ll immunotherapy alone had different antitumor effects depending on the tumor model, with 4T1 and CT26 transplants being resistant to anti-PD-Ll MoAb, whereas E0771 mouse transplants were sensitive to anti-PD-Ll therapy, which also resulted in mild prolongation of animal survival when used as a single therapy. Notably, combining oral Leucine supplementation with anti-PD-Ll immunotherapy cooperatively slowed down in vivo tumor growth and prolonged animal survival; in addition, in some animals treated with this combination completed disappearance of the primary tumor mass was observed, with some mice also achieving long-term survival. Finally, in subcutaneous LLC model, which is also resistant to anti-PDLl MoAb when used alone but it becomes sensitive when MoAb is combined with fasting4, combining Leu+fasting with anti-PD-Ll significantly slowed down tumor growth and prolonged animal survival when compared to anti-PDLl MoAb + fasting.

[0133] Together, these results indicate that oral Leucine supplementation can sensitize immunotherapyresistant tumors to the effects of anti-PD-Ll immunotherapy; at the same time, Leucine supplementation significantly enhances the antitumor effects of anti-PD-Ll MoAb in immunotherapy-sensitive malignancies. Of note, animals treated with the combination of oral Leucine and immunotherapy did not show any sign of suffering, thus suggesting that these combinations result in enhanced antitumor effects without causing severe adverse events.

[0134] Due to the widespread use of anti-PDl or anti-PD-Ll immune checkpoint inhibitors in patients with both early stage and advanced TNBC, CRC or lung carcinomas, our findings indicate that oral Leucine supplementation is a promising therapeutic strategy to boost the antitumor activity and efficacy of anti-PDl or anti-PD-Ll MoAb regardless of primary tumor sensitivity / resistance to immunotherapy.

[0135] Leucine supplementation enhances antitumor activity and efficacy of chemotherapy in TNBC, CRC and lung cancers models

[0136] Despite the recent advent of new effective treatments, such as immunotherapy or targeted therapies, cytotoxic chemotherapy, including platinum (i.e., cisplatin, carboplatin or oxaliplatin) or taxane (paclitaxel, docetaxel, nab-paclitaxel)-based chemotherapy remains the most effective, standard- of-care treatment option for patients with early-stage or advanced malignancies, including TNBC and CRC.

[0137] Here, it was investigated if oral Leucine supplementation cooperates with platinum / taxane chemotherapy in syngeneic models of TNBC (orthotopic 4T1 or E0771 transplants in female BALB / c and C57BL6 / J mice, respectively) and in subcutaneous models of CRC and lung cancer. As shown in Figure 3, weekly intraperitoneal (i.p.) injection of carboplatin (4T1 and LLC), paclitaxel (E0771) or oxaliplatin (CT26) slowed down in vivo tumor growth and resulted in statistically significant prolongation of animal survival, with the therapeutic effect being different according to the tumor type (i.e., more evident in E0771 transplants, and less evident in 4T1, LLC and CT26 transplants).

[0138] Of note, oral Leucine supplementation cooperated with chemotherapy in slowing down in vivo tumor growth and in prolonging animal survival. Again, the most relevant effects of combination treatment (Leu plus chemotherapy), in terms of reduced primary tumor growth progression and animal survival prolongation, was observed in mouse E0771 transplants, while it was less evident, yet still statistically significant, in 4T1, LLC and CT26 mouse transplants.

[0139] Together, the results indicate that oral Leucine supplementation enhances the antitumor activity and efficacy of platinum- andtaxane-based chemotherapy in highly aggressive and rapidly deadly mouse models of TNBC and CRC.

[0140] Combining oral Leucine supplementation with chemo-immunotherapy and fasting results in long-term survival and in some animal cures in highly aggressive TNBC and CRC mouse models The goal of the current preclinical and clinical research in oncology is to prolong the survival of patients with highly aggressive malignancies and, eventually, to cure some of these patients. However, this is a difficult goal to achieve, mainly as a result of tumor heterogeneity and to the difficulty to eradicate all tumor cells.

[0141] Here, it was investigated whether combining chemo-immunotherapy (namely, platinum- or taxane- based chemotherapy plus anti-PD-Ll MoAb), which is now the standard-of-care treatment in several patients with advanced malignancies, including advanced TNBC and lung carcinoma patients, with oral Leucine plus cyclic fasting (an experimental antitumor intervention that showed promising antitumor effects - see Figure 1), can result in long-term animal survival.

[0142] For these experiments, the inventors used the same TNBC (4T1, E0071) and CRC (CT26) and lung cancer (LLC) mouse models used in previous experiments. As shown in Figure 4, combining chemo-immunotherapy with oral Leucine (daily 500 mg / kg Leucine by oral gavage) plus cyclic fasting (48-hour water-only fasting followed by ad libitum refeeding, and repeated every 7 days) results in meaningful prolongation of animal survival when compared to the fasting-Leucine doublet. In addition, and even more importantly in terms of potential translational implications, the quadruple treatments resulted in complete regression of the primary tumor in some mice bearing E0771 or CT26 cell transplants, and some animals were still alive some months after the initiation of these treatments.

[0143] Together, these data suggest that the combination of an experimental therapy, such as oral Leucine supplementation plus cyclic fasting / FMD, with standard chemoimmunotherapy combinations can result in long-term animal survival and animal cure in highly aggressive and deadly models of TNBC, CRC and lung cancer.

[0144] Fasting plus Leu supplementation enhances chemoimmunotherapy antitumor activity

[0145] Chemoimmunotherapy (ChT-IO) combinations have become the standard-of-care treatment of several human malignancies, including early-stage and advanced TNBC and lung carcinoma 13,15,18,20 Teinvenqonprovides Leu supplementation plus cyclic water-only fasting or FMD as a novel, safe and highly effective metabolic intervention that could be easily translated in the clinic to boost the immunomodulatory and anticancer activity of standard ChT-IO combinations. The inventors studied the antitumor effects of combining ChT-IO with the Leu-fasting doublet in TNBC (Fig 5 A-D), CRC (Fig 5 E-F) and lung carcinoma (Fig 5 G-H) models, with the cytotoxic drug varying according to the tumor type. In all these models, the quadruple treatments were well tolerated, and they did not cause toxic deaths nor excessive weight loss when compared to double treatments. Of note, ChT-IO plus Fasting+Leu combinations remarkably slowed down tumor progression and prolonged animal survival when compared to double treatments (ChT-IO or Fasting+Leu). In highly aggressive E0771 and CT26 tumors, the quadruple treatments cured 40% and 30% of animals, respectively.

[0146] Different daily dosages of oral Leu had the same antitumor efficacy in combination with fasting and ChT-IO in terms of in vivo tumor growth delay and animal survival prolongation, which indicates a plateau in the dose-response curve of Leu-based pharmacologic combinations (Fig 5 I- J). In terms of metabolic changes, ChT-IO combinations mildly increased blood glucose and insulin levels when compared to CTRL conditions, whereas Fasting+Leu, alone or in combination with ChT-IO, significantly reduced blood glucose and insulin concentration when compared to ChT-IO alone (Fig 5 K-L).

[0147] Leu anticancer effects are mediated by CD8+ T lymphocytes

[0148] To confirm the lack of tumor cell autonomous Leu antitumor effects in vivo, 4T1 cells were injected into NOD-scid IL2rgnull (NSG) mice, which lack both T cells and fully functional NK cells. Animals were randomly assigned to CTRL, cyclic fasting, Leu supplementation, or fasting + Leu combination (Figure 6 A, B). Interestingly, Leu supplementation did not affect tumor progression, both when used alone nor when combined with fasting. Similarly, Leu supplementation did not delay primary tumor growth in E0771-bearing NSG mice (Figure 6 C, D). These data indicate that the antitumor effects of Leu are not tumor-cell autonomous and they require an intact immune system.

[0149] To study whether Leu supplementation boosts CD8+ T lymphocyte activity, the specific role of CD8+ T cells in mediating Leu antitumor effects was investigated. To this aim, CD8+ T cells were depleted through an anti-CD8 murine monoclonal antibody (MoAb) before randomly assigning 4Tl-bearing BALB / c mice to different groups. As a control for potential off-target effects of the MoAb, non-specific murine IgG were used. Leu supplementation slowed down tumor growth and prolonged animal survival when used alone and showed cooperative anticancer activity when combined with cyclic fasting in mice treated with control IgG (Figure 6 E, F). Notably, Leu supplementation, alone or when combined with fasting, was no longer effective in mice treated with anti-CD8 MoAb (i.e., undergoing CD8+ T cell depletion), thus indicating that CD8+ T cells are essential for Leu antitumor effects (Figure 6 G, H).

[0150] To investigate whether CD8+ T cells are also sufficient to promote Leu antitumor activity, CD8+ T cells were collected from the spleen and lymph nodes of untreated healthy C57BL / 6J mice, and they were injected intravenously in E0771 -bearing NSG mice which were subsequently exposed to CTRL, Leu, or Leu + fasting conditions. CD8+ T cell transfer to E0771-bearing NSG mice sensitized tumors to the treatment with Leu, alone or in combination with fasting (Figure 6 I, J).

[0151] An excess of Leu primes tumor cells to be recognized by CD8+ T lymphocytes

[0152] Having established CD8+ T cell role in mediating Leu antitumor effects, it was investigated whether Leu directly acts on CD8+ T cells to boost their cytotoxic / memory functions or if Leu primes cancer cells to be subsequently recognized by lymphocytes. To address the former point, firstly CD8+ T cells were collected from spleen and lymph nodes of healthy C57 / BL6J mice previously exposed to CTRL, Leu or Leu + fasting combination. Then, isolated CD8+ T cells were injected into untreated E0771 -bearing NSG mice. The transfer of CD8+ T cells previously exposed to Leu or fasting + Leu did not affect the progression of E0771 tumors in NSG mice when compared to the transfer of CD8+ T cells collected from animals not exposed to Leu (Figure 7 A, B). These results indicate that Leu does not directly act on CD8+ T cells to promote their activation. Alternatively, Leu may prime tumor cells to be recognized by lymphocytes. To test this hypothesis, CD8+ T cells were collected from healthy C57 / BL6J mice, and injected into E0771-bearing NSG mice that were exposed to Leu or fasting + Leu only before CD8+ T cells transfer, thus totally avoiding the exposure of injected CD8+ T cells to Leu. Interestingly, and in line with the underlying hypothesis of the invention, CD8+ T cell injection significantly delayed tumor progression and improved survival of NSG mice previously treated with Leu or fasting + Leu when compared to not treated mice (Figure 7 C, D).

[0153] To confirm that Leu primarily acts on cancer cells to retard tumor progression, 4T1 cells engineered to stably overexpress Branched Chain Amino Transferase 1 (BCAT1), a cytoplasmic enzyme that catalyzes the first biochemical reaction of the BCAA degradation pathway, were used. 4T1 cells engineered with an empty plasmid (blank) were used as a control. BCAT1-0E 4T1 cells degrade Leu more efficiently and prevent intratumor Leu accumulation without affecting plasma Leu levels upon Leu supplementation. BCAT1-0E 4T1 or control cells (blank) were injected in the mammary fat pad of female BALB / c mice, which were exposed to the following treatment conditions: CTRL, Leu, fasting, or fasting + Leu. While Leu and fasting + Leu combination delayed tumor progression and prolonged animal survival when compared to CTRL and fasting conditions, respectively, in mice injected with control cells (blank) (Figure 7 E, F), BCAT1-0E tumors were resistant to Leu supplementation plus / minus fasting (Figure 7 G, H). Since in this experimental system Leu is specifically degraded in BCAT1-0E tumor cells (and not in normal murine cells, including CD8+ T cells), it is possible to conclude that an increased concentration of Leu in cancer cells is essential for Leu-induced antitumor effects. Together, these experiments indicate that Leu primes tumor cells to be recognized by CD8+ T cells, which acquire an activated phenotype when they encounter tumor cells that were previously exposed to Leu; on the other hand, direct lymphocyte exposure to Leu is not essential to mediate these antitumor effects.

[0154] It was further investigated whether the acquisition of a memory phenotype plays an important role in the antitumor effects of Leu supplementation. To this aim, CD8+ T cells were collected from the spleen and lymph nodes of E0771-bearing C57BL / 6J mice exposed to CTRL, Leu or Leu + fasting for 11 days (2 fasting cycles). Collected CD8+ T cells were injected in the tail vein of untreated NSG mice 5 days after E0771 cell implantation in the mammary fat pad. The transfer of CD8+ T cells collected from mice exposed to Leu and fasting + Leu delayed tumor progression and prolonged animal survival when compared to the transfer of CD8+ T cells collected from untreated CTRL mice (Figure 7 I, J). This result suggests that the antitumor phenotype acquired by CD8+ T cells after 11 days of Leu exposure is maintained also when these cells are transplanted into untreated mice, thus indicating that short tumor exposure to Leu is sufficient to prime tumor cells to be recognized by CD8+ T cells, and also sufficient to allow lymphocytes establish a memory phenotype that is maintained.

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Claims

CLAIMS1. Leucine or a pharmaceutically acceptable salt thereof for use in the treatment of cancer either alone or in combination with at least one water-only fasting cycle or with a fasting mimicking diet and / or a further therapeutic intervention.

2. Leucine or the pharmaceutically acceptable salt thereof for use in the treatment of cancer according to claim 1 wherein said at least one water-only fasting cycle is characterized by 24 h to 72 h, preferably 24 h to 48 h, preferably 48 h of water-only 100% caloric restriction.

3. Leucine or the pharmaceutically acceptable salt thereof for use according to claim 1, wherein said fasting mimicking diet lasts for a period between 24 and 190 hours, preferably said fasting mimicking diet lasts for a period of 24 to 150 hours, preferably said fasting mimicking diet lasts for approximately 120 hours.

4. Leucine or the pharmaceutically acceptable salt thereof for use according to claim 3, wherein said fasting mimicking diet is a regular caloric intake reduced by 65% to 85% and / or said fasting mimicking diet is based on an average caloric intake of between 200 and 1200 Kcal / day, preferably between 300 and 600 Kcal / day.

5. Leucine or the pharmaceutically acceptable salt thereof for use according to claim 4, wherein said fasting mimicking diet comprises a first period of 0 to 24 hours wherein the caloric intake is a regular caloric intake reduced by approximately 65%, followed by a second period of 24 to 96 hours wherein the caloric intake is a regular caloric intake reduced by approximately 85%, preferably said first period lasts approximately 24 hours and said second period lasts approximately from 48 to 96 hours.

6. Leucine or the pharmaceutically acceptable salt thereof for use according to any one of claims 3 to 5, wherein said fasting mimicking diet comprises a reduced protein intake and / or a reduced simple carbohydrate intake.

7. Leucine or the pharmaceutically acceptable salt thereof for use according to anyone of claims 3 to 6, wherein said fasting mimicking diet comprises administering a food having a content of monounsaturated and / or polyunsaturated fats between 10% and 80 % of the total calorie amount, a content of proteins from 0 to 10% of the total calorie amount, and a content of carbohydrates from 5% to 50% of the total calorie amount.

8. Leucine or the pharmaceutically acceptable salt thereof for use according to any one of previous claims, wherein said leucine is administered in an amount of approximately between lOOmg / kgand lOOOmg / kg on a daily basis, preferably between 150 mg / kg and 800mg / kg on a daily basis, preferably between 250 mg / kg and 600 mg / kg on a daily basis.

9. Leucine or the pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 7 , wherein said leucine is administered in an amount of approximately between 10 mg / kg and 1000 mg / kg on a daily basis, preferably between 25 mg / kg and 800 mg / kg on a daily basis, preferably between 50 mg / kg and 500 mg / kg on a daily basis.

10. Leucine or the pharmaceutically acceptable salt thereof for use according to any one of previous claims, wherein said further therapeutic intervention is selected from the group consisting of: surgery, radiotherapy and a further therapeutic agent.

11. Leucine or the pharmaceutically acceptable salt thereof for use according to claim 10, wherein said further therapeutic agent is an immune checkpoint inhibitor (ICI), preferably a PD-1 inhibitor, and / or PDL-1 inhibitor and / or CTLA-4 inhibitor.

12. Leucine or the pharmaceutically acceptable salt thereof for use according to claim 10, wherein said further therapeutic agent is a chemotherapeutic agent, preferably said chemotherapeutic agent is selected from the group consisting of: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an antibody-drug conjugate (ADC) delivering a topoisomerase inhibitor or a microtubule poison, preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab- paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide, trastuzumab-deruxtecan, sacituzumab-govitecan, datopotamab-deruxtecan, sacituzumab- tirumotecan, enfortumab-vedotin, trastuzumab emtansine (T-DM1).

13. Leucine or the pharmaceutically acceptable salt thereof for use according to claim 10, wherein said further therapeutic agent is a combination of chemotherapeutic agent and an immunotherapy agent, preferably said chemotherapeutic agent is selected from the group consisting of: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an ADC delivering a topoisomerase inhibitor or a microtubule poison, preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab-paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclophosphamide; and the immunotherapy agent is an immune checkpoint inhibitor (ICI), preferably a PD-1 inhibitor, and / or PDL-1 inhibitor and / or CTLA-4 inhibitor.

14. Leucine or the pharmaceutically acceptable salt thereof for use according to claims 10 or 13, wherein said further therapeutic agent is a combination of carboplatin or paclitaxel or nab- paclitaxel or oxaliplatin and a PD-1 inhibitor.

15. Leucine or the pharmaceutically acceptable salt thereof for use according to any one of previous claims, wherein said cancer is selected from the group consisting of: breast cancer, colorectal cancer, small cell and non small cell lung carcinomas (SCLCs and NSCLCs), pancreatic adenocarcinoma, colon cancer, rectal cancer, mucinous adenocarcinoma, melanoma, biliary tract carcinoma, gastric carcinoma, esophageal carcinoma, small intestine carcinoma, urothelial carcinoma, neuroblastomas, gliomas, sarcomas, lymphomas, leukemias, neuroendocrine carcinomas, either early stage or metastatic.

16. Leucine or the pharmaceutically acceptable salt thereof for use according to any one of previous claims, wherein said cancer is resistant to conventional therapy, preferably said cancer is resistant to radiotherapy or chemotherapy, preferably said cancer is resistant to: a DNA synthesis inhibitor, a DNA damaging agent, a topoisomerase inhibitor, a microtubule poison, an ADC delivering a topoisomerase inhibitor or a microtubule poison, preferably said chemotherapeutic agent is selected from the group consisting of: oxaliplatin, carboplatin, cisplatin, paclitaxel, docetaxel, nab- paclitaxel, eribulin, doxorubicin, epirubicin, irinotecan, gemcitabine, cyclopshosphamide, trastuzumab-deruxtecan, sacituzumab-govitecan, datopotamab-deruxtecan, sacituzumab- tirumotecan, enfortumab-vedotin, trastuzumab emtansine (T-DM1).

17. A pharmaceutical or nutritional composition or dietary supplement comprising leucine or the pharmaceutically acceptable salt thereof for use according to any one of previous claims, and a pharmaceutical or nutritionally acceptable carrier.

18. The pharmaceutical or nutritional composition or dietary supplement for use according to claim 17 formulated as an oral formulation with delayed or prolonged intestinal absorption.

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