Combination of extracellular vesicles and chemotherapeutic agent and uses thereof for the treatment of cancers such as peritoneal carcinomatosis

Abstract

Several gastrointestinal and gynecological malignancies have the potential to disseminate and grow in the peritoneal cavity. The occurrence of peritoneal carcinomatosis (PC) has been shown to significantly decrease overall survival in patients. Treatment of residual microscopic disease remains a challenge with new anticancer modalities development. Now, the inventors propose an innovative therapeutic management of peritoneal carcinomatosis (PC) that is bio-inspired and tumor-targeted by combining MSC-derived EVs and at least one chemotherapeutic agent for improved chemotherapy efficiency ty. In this work, the present inventors have shown in two different mice models of PC and with two different chemotherapeutic agents that, while the MSC-derived EVs from mice (mEVs) have no effect on mice suffering from PC, treatment with mEvs combined with chemotherapeutic agent has a superior therapeutic effect i.e. a significant reduction of the Peritonal Carcinomatosis Index (PCI) compared to treatment with chemotherapeutic agent alone. Thus, the present invention relates to the combination of EVs and at least one chemotherapeutic agent and uses thereof for the treatment of cancers such as PC.

Description

[0001] COMBINATION OF EXTRACELLULAR VESICLES AND CHEMOTHERAPEUTIC AGENT AND USES THEREOF FOR THE TREATMENT OF CANCERS SUCH AS PERITONEAL CARCINOMATOSIS

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine and in particular in the field of oncology.

[0004] BACKGROUND OF THE INVENTION:

[0005] Peritoneal carcinomatosis (PC) describes the dissemination of cancer deposits within the peritoneal cavity. The therapeutic modalities and prognosis depend on the origin of the primary cancers. Numerous cancers can produce peritoneal carcinomatosis including cancers of the endometrium, fallopian tubes, ovaries, uterus, colon, rectum, small bowel, gall bladder, bile duct, appendix, stomach, pancreas, liver and breast. Peritoneal carcinomatosis arising from local spread of abdominal tumors is poorly responsive to systemic chemotherapy. For example, peritoneal carcinomatosis secondary to colorectal cancer have a poor prognosis, about 16 months with chemotherapy treatment.

[0006] Amongst the curative and preventive treatments of peritoneal carcinomatosis, one can cite intra-abdominal chemotherapy, carried out by irrigation of the peritoneum at normal temperature and without increased pressure.

[0007] An alternative method of treating peritoneal carcinomatosis is hyperthermic intraperitoneal chemotherapy (HIPEC) which consists in using a chemotherapeutic agent warmed to a temperature comprised between 38.5°C and 46.0°C.

[0008] The development of combined treatment involving cytoreductive surgery (CRS) and HIPEC permitted to improve survival of patients suffering from peritoneal carcinomatosis secondary to colorectal cancer [Elias et al, 2009, J. Clin. Oncol., 27(5):681-685 ; Elias et al, 2010, Eur. J. Surg. Oncol., 36(5):456-462 ; Sugarbaker et al, 2012, Gastroenterol. Res. Pract., 623417] with a median survival of more than 40 months [Elias et al, 2009, J. Clin. Oncol., 27(5):681-685 ; Elias et al, 2010, Eur. J. Surg. Oncol., 36(5):456-462 ; Chua et al, 2009, Ann. Surg., 249(6) :900-907 ;Quenet et al, 2011 Ann. Surg., 254(2):294-301]. However the recent result of the first phase III trial (PRODIGE 7) comparing CRS alone with CRS combined with HIPEC using oxaliplatin, failed to demonstrate an overall survival advantage in the HIPEC arm [Quenet et al, 2018, J. Clin. Oncol., 36(18), Meeting Abstract ASCO Annual Meeting II; Ceelen, 2019, Eur. J. Surg. Oncol., 45(3):400-402]. Post-operative morbidity rate, with a 60 day complication rate significantly higher (24.1% vs 13.6%, p=0.03) for patients treated with HIPEC [Quenet et al, 2018, J. Clin. Oncol., 36(18) suppl: Meeting Abstract ASCO Annual Meeting II ; Ceelen, 2019, Eur. J. Surg. Oncol., 45(3):400-402] could explain the lack of benefit in the PRODIGE 7 trial. Treatment of residual microscopic disease remains a challenge with new anticancer modalities development.

[0009] Bio-camouflaged and bio-inspired approaches are currently very attractive strategies in the emerging nanomedicine landscape for the delivery of drugs. In particular, extracellular vesicles (EVs) could represent a drug delivery vehicle of choice considering their endogenous properties of stability in blood circulation, immunotolerance and capacity to facilitate cell entry. EVs are considered to be natural nanocarriers with improved biocompatibility and intrinsic bioactivity (originating from parental cells), which is advantageous for clinical application. Similarly to liposomes, the EV membrane is essentially a lipid bilayer protecting the payload from degradation, but with natural targeting properties and relative stability in blood circulation.

[0010] In the specific context of tumor therapy, mesenchymal stem cells (MSCs) present inherent tumor-trophic properties whose MSC-derived EVs could inherit, which allow them to serve as vehicles for targeted therapy to isolated tumors and metastatic disease. Contrary to synthetic nanovectors such as liposomes, MSC- derived EVs appear like a biogenic drug delivery vehicles, to deliver therapeutic agents such as therapeutic miRNA and anti-cancer agents [Silva et al, 2015, Sci. Rep., 5:7763 ; Moore et al, 2017, Int. J. Cancer, 141(3):428-436 ; Chulpanova et al, 2018, Front. Immunol., 9:1534]. In addition, MSC-derived EVs present inherent bioactive properties, stemming from their parental cell inherited cargo, that can for example modulate the tumor immune microenvironment (Pinto et al, 2021, ACS Nano., 15(2):3251-3263).

[0011] From this perspective, the present inventors have already proposed a therapeutic management of PC that is bio-inspired and tumor-targeted by engineering MSC-derived EVs to encapsulate a photosensitizer such as temoporfin (mTHPC) for improved photodynamic therapy efficiency and safety (International application WO 2021 / 099575 Al). This treatment method necessitates preparing MSC-derived EVs loading or encapsulating photosensitizers.

[0012] The present inventors aim to identify additional compositions and methods for preventing and / or treating PC with improved properties.

[0013] SUMMARY OF THE INVENTION:

[0014] As defined by the claims, the present invention relates to a combination of extracellular vesicles such as mesenchymal stem cell-derived extracellular vesicles and chemotherapeutic agents and use thereof for the prevention and / or the treatment of cancers, in particular cancers occurring in body cavity such as peritoneal carcinomatosis.

[0015] DETAILED DESCRIPTION OF THE INVENTION:

[0016] The present invention enables the purpose set by the inventor to be reached.

[0017] Indeed, the present inventors have surprisingly found that co-administering mesenchymal stem cell-derived extracellular vesicles and chemotherapeutic agent could significantly improve the therapeutic effect of this chemotherapeutic agent. More particularly, the present inventors have shown in two different mice models of peritoneal carcinomatosis (PC) which are mice injected with CT26 cells or MC38 cells and with two different chemotherapeutic agents (mitomycin and oxaliplatin) that, while the MSC-derived EVs from mice (mEVs) have no effect on mice suffering from PC, treatment with mEVs combined with chemotherapeutic agent has a superior therapeutic effect i.e. a significant reduction of the Peritonal Carcinomatosis Index (PCI) compared to treatment with chemotherapeutic agent alone.

[0018] At the time of the present invention, the prior art teaching would have disclosed and / or suggested to the one skilled in the art to load the mesenchymal stem cell-derived extracellular vesicles with chemotherapeutic agent and nothing would have suggested to the one skilled in the art that a surprising therapeutic effect could be obtained simply by combining chemotherapeutic agents with mesenchymal stem cell-derived extracellular vesicles.

[0019] Since the present invention does not require the mesenchymal stem cell- derived extracellular vesicles to be loaded with chemotherapeutic agents, the treatment method according to the present invention is simplified compared to prior art treatment methods.

[0020] In addition, the surprising effect observed for PC and using mesenchymal stem cell derived extracellular vesicles can be helpful in any kind of cancers and in particular in any cancer occurring in a body cavity and using any extracellular vesicles.

[0021] Thus, the first object of the present invention relates to a combination of extracellular vesicles, such as mesenchymal stem cell-derived extracellular vesicles, and of at least one chemotherapeutic agent.

[0022] The combination of extracellular vesicles, such as mesenchymal stem cell- derived extracellular vesicles and of at least one chemotherapeutic agent according to the present invention can also be defined as a kit of parts of extracellular vesicles such as mesenchymal stem cell derived extracellular vesicles and of at least one chemotherapeutic agent.

[0023] It should be emphasized that, in the combination according to the present invention, the chemotherapeutic agent(s) is / are not loaded, introduced or inserted into or onto the EVs such as MSC-EVs of this combination.

[0024] The combination according to the present invention is helpful for coadministration of EVs such as MSC-derived EVs and chemotherapeutic agent(s). As used herein, the expression “extracellular vesicle” refers to a cell- derived vesicle comprising a membrane that encloses an internal space. Extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally, extracellular vesicles range in diameter from 50 nm to 1000 nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. EVs can derive from any prokaryotic or eukaryotic cell and in particular from any mammalian cell, any plant cell and any micro-organism.

[0025] As used, herein, the expressions “mesenchymal stem cells”, “mesenchymal stem / stromal cells” or “MSCs” has its general meaning in the art and refers to multipotent stromal cells that can differentiate into a variety of cell types, including: osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells) (See, for example, Wang, 2004, Stem Cells, 22(7): 1330- 1337 ; McElreavey, 1991, Biochem. Soc. Trans., (l);29s ; Takechi, 1993, Placenta, 14(2):235-245 ; Kobayashi, 1998, Early Human Development, 51(3):223-233 ; Yen, 2005, Stem Cells, 23(l):3-9) .These cells may be defined phenotypically by gene or protein expression. These cells have been characterized to express (and thus be positive for) one or more of CD13, CD29, CD44, CD49a, b, c, e, f, CD51, CD54, CD58, CD71, CD73, CD90, CD 102, CD105, CD106, CDwll9, CD120a, CD120b, CD123, CD124, CD126, CD127, CD140a, CD166, P75, TGF-blR, TGF-bllR, HLA-A, B, C, SSEA-3, SSEA-4, D7 and PD-L1. These cells have also been characterized as not expressing (and thus being negative for) CD3, CD5, CD6, CD9, CD10, CDl la, CD14, CD15, CD18, CD21, CD25, CD31, CD34, CD36, CD38, CD45, CD49d, CD50, CD62E, L, S, CD80, CD86, CD95, CD117, CD133, SSEA-1, and ABO. MSCs can be isolated using methods known in the art, e.g., from bone marrow mononuclear cells, umbilical cord blood, adipose tissue, placental tissue, based on their adherence to tissue culture plastic. For example, MSCs can be isolated from commercially available bone marrow aspirates. Enrichment of MSCs within a population of cells can be achieved using methods known in the art including but not limited to FACS.

[0026] Thus, as used herein, the expressions “mesenchymal stem cell-derived extracellular vesicle”, “mesenchymal stem / stromal cell-derived extracellular vesicle”, “MSC-derived EV” or “MSC-EV” are equivalent and can be used interchangeably. They refer to an extracellular vesicle originated from mesenchymal stem cells. Thus, the MSC-derived EVs implemented in the present invention are identifiable by the detectable presence of a particular surface epitope or combination of surface epitopes, and / or by the absence of particular surface epitopes. For example, the MSC-derived EVs implemented in the invention are characterized by the presence of vesicle surface detectable levels of the epitope CD63 (i.e., CD63(+)), CD81 (i.e., CD81+), and CD9 (i.e., CD9+). In some embodiments, the diameter of the MSC-derived EVs implemented in the present invention is about 175 nm.

[0027] In the combination according to the present invention, the EVs such as MSC-derived EVs implemented are isolated and / or highly purified.

[0028] As used herein, the terms “isolated”, “isolating”, “purified”, "purifying," "enriched," and "enriching," as used herein with respect to cells, means that the EVs such as MSC-derived EVs at some point in time were separated, purified and capable of therapeutic use. "Highly purified", “highly enriched”, and “highly isolated” when used with respect to said extracellular vesicles, indicates that the cells of interest are at least about 70%, about 75%, about 80%, about 85% about 90% or more of the cells, about 95%, at least 99% pure, at least 99.5% pure, or at least 99.9% pure or more of the cells, and can preferably be about 95% or more of the EVs.

[0029] In some embodiments, the EVs such as MSC-derived EVs implemented in the present invention are prepared by any method well known in the art. In some embodiments, the EVs such as MSC-derived EVs of the present invention are prepared by methods for 3D culture that are well known in the art, and include, but are not limited to standard culture in 2D flasks, hanging drop culture, culturing on matrices, culturing on microcarriers, culturing on synthetic extracellular scaffolds, culturing on chitosan membranes, culturing under magnetic levitation, suspension culture in rotating bioreactors, or culturing under non-contact inhibition conditions. See, e.g., Haycock, 2011, Methods Mol Biol., 695:1-15 ; Lee et al, 2008, Tissue Eng. Part B Rev., 14(l):61-86 ; Pampaloni et al, 2007, Nat. Rev. Mol. Cell Biol., 8(10):839-45 ; and Souza et al, 2010, Nat. Nanotechnol., 5(4):291-296.

[0030] In some embodiments, the EVs such as MSC-derived EVs implemented in the present invention are prepared by the system culture described in the International application WO 2019 / 002608 Al. In particular, the EVs such as MSC-derived EVs implemented the present invention are prepared according to the method described in experimental part of the International application WO 2021 / 099575 Al. More particularly, the method involves a fluid system comprising at least one container, a liquid medium contained by the container and producer cells, characterized in that it also comprises microcarriers suspended in the liquid medium, the majority of the producer cells being adherent to the surface of the microcarriers, and a liquid medium agitator, the agitator and the dimensions of the container being capable of controlling a turbulent flow of the liquid medium in the container.

[0031] Advantageously, the cells and in particular the mesenchymal stem cells from which are derived the EVs and in particular the MSC-derived EVs implemented in the present invention come from or originate from the same species as the subject to be treated using the combination of the present invention.

[0032] As used herein, the expression “chemotherapeutic agent” refers to any compound that can be used in the treatment, management or amelioration of cancer, including peritoneal carcinomatosis, or the amelioration or relief of one or more symptoms of a cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclo sphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiopho sphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, authramycin, azascrine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycins, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhy dr azide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vinblastine; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; difhioromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine; imexon; tyrosine kinase inhibitors, such as epidermal growth factor receptor tyrosine kinase inhibitor erlotinib; checkpoint inhibitors and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0033] As used herein, the expression “checkpoint inhibitors” refer to a type of immunotherapeutic agents useful for treating cancers. These antibody-based inhibitors block different checkpoint proteins such as cytotoxic T-lymphocyte- associated protein 4 (or CTLA-4), programmed cell death 1 receptor (PD-1) and programmed cell death receptor ligand 1 (PDL-1). The checkpoint inhibitors useful in the present invention can be CTLA-4 inhibitors such Ipilimumab and Tremelimumab; PD-1 inhibitors such as Cemiplimab, Nivolumab and Pembrolizumab; and PD-L1 inhibitors such as Atezolizumab, Durvalumab and Avelumab.

[0034] In some embodiments, the chemotherapeutic agent(s) implemented in the present invention is / are selected in the group consisting of capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, epirubicin, erlotinib, 5-fluorouracil, gemcitabine, irinotecan, leucovorin, oxaliplatin, paclitaxel, topotecan, checkpoint inhibitors and any mixture thereof.

[0035] In some embodiments, the chemotherapeutic agent(s) implemented in the present invention is / are selected in the group consisting of cisplatin, doxorubicin, oxaliplatin, CTLA-4 inhibitors such Ipilimumab and Tremelimumab, PD-1 inhibitors such as Cemiplimab, Nivolumab and Pembrolizumab, and PD-L1 inhibitors such as Atezolizumab, Durvalumab and Avelumab and any mixture thereof.

[0036] In the combination according to the present invention, the EVs such as MSC-derived EVs and the at least one chemotherapeutic agent may be formulated as two pharmaceutical compositions. Thus, in the combination according to the present invention, the EVs such as the MSC-derived EVs may be in a first pharmaceutical composition and the at least one chemotherapeutic agent in a second pharmaceutical composition. The first pharmaceutical composition of the combination according to the present invention comprises, as active ingredient, EVs such as MSC-derived EVs and at least one pharmaceutically acceptable vehicle.

[0037] The second pharmaceutical composition of the combination according to the present invention comprises, as active ingredient, at least one chemotherapeutic agent and at least one pharmaceutically acceptable vehicle.

[0038] In some embodiments, the pharmaceutically acceptable vehicle of the first pharmaceutical composition may be identical to the one of the second pharmaceutical composition. Alternatively, the pharmaceutically acceptable vehicle of the first pharmaceutical composition may be different from the one of the second pharmaceutical composition.

[0039] The expression “pharmaceutically acceptable vehicle” as used herein is meant any substance which is added to either EVs such as MSC-derived EVs or to chemotherapeutic agent to promote their or its transport, avoid their or its substantial degradation in the pharmaceutical composition and / or increase their or its half-life. Advantageously, such a pharmaceutically acceptable vehicle is sterile and non-pyrogenic and refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, in particular, a mammal, especially a human, as appropriate.

[0040] It is chosen depending on the type of application of the pharmaceutical compositions implemented in the invention and in particular as a function of its administration mode. Advantageously, a pharmaceutically acceptable vehicle refers to a non-toxic, solid, semi-solid or liquid carrier, filler, diluent, additive, excipient, buffer, encapsulating material or formulation auxiliary of any type.

[0041] In some embodiments, the combination and consequently the first pharmaceutical composition and the second pharmaceutical composition are parenterally administrated. Thus the first and the second pharmaceutical compositions for parenteral administration implemented in the invention can be sterilized, aqueous or non-aqueous solution, suspension or emulsion. As a solvent or vehicle, water, propylene-glycol, polyethylene glycol, plant oils, injectable ester like ethyl oleate or other suitable organic solvents can be used. These compositions can also contain adjuvants, such as wetting agents, isotonising agents or emulsifiers.

[0042] The combination according to the present invention is a therapeutically effective combination.

[0043] As used herein, the term “therapeutically effective combination” as used herein refers to an amount or dose of EVs such as MSC-derived EVs together with the amount or dose of the chemotherapeutic agent(s) that is sufficient to prevent cancer, in particular, a cancer occurring in a body cavity and, more particularly, peritoneal carcinomatosis.

[0044] The amount of the EVs such as MSC-derived EVs and / or of the chemotherapeutic agent(s) in a given therapeutically effective combination may be different for different individuals and different tumor types, and will be dependent upon the one or more additional agents or treatments included in the combination.

[0045] The "therapeutically effective amount" is determined using procedures routinely employed by those skilled in the art such that an “improved therapeutic outcome” results. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.

[0046] The specific therapeutically effective dose level for the combination, the EVs such as MSC-derived EVs or the chemotherapeutic agent(s) will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the EVs such as the MSC-derived EVs and the chemotherapeutic agent(s) employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the EVs such as the MSC-derived EVs and of the chemotherapeutic agent(s) at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. In the combination according to the present invention, the first pharmaceutical composition contains between 108and 1013EVs such as the MSC- derived EVs and particularly between 108and 1012EVs such as the MSC-derived EVs / ml of this composition.

[0047] In addition, the daily dosage of the chemotherapeutic agent(s) as implemented in the present invention may be varied over a wide range from 0.01 to 1,000 mg per adult per day. The combination according to the present invention and, in particular, the second pharmaceutical composition contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the chemotherapeutic agent(s) for the symptomatic adjustment of the dosage to the subject to be treated. In the combination according to the present invention, the second pharmaceutical composition contains from about 0.01 mg to about 500 mg of the chemotherapeutic agent(s) and, in particular, from 1 mg to about 100 mg of the chemotherapeutic agent(s) / ml of this composition. An effective amount of the chemotherapeutic agent(s) is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0048] In another aspect, the present invention relates to the combination as previously defined for use in treating cancers.

[0049] Thus, the present invention concerns

[0050] - in one hand, chemotherapeutic agent(s) or a second pharmaceutical composition as previously defined for use in treating cancers, wherein said chemotherapeutic agent(s) is / are co-administered to a subject in need thereof with extracellular vesicles such as mesenchymal stem cell-derived extracellular vesicles or a first pharmaceutical composition as previously defined and

[0051] - in the other hand, extracellular vesicles such as mesenchymal stem cell- derived extracellular vesicles or a first pharmaceutical composition as previously defined for use in treating cancers, wherein said extracellular vesicles such as mesenchymal stem cell-derived extracellular vesicles or said first pharmaceutical composition as previously defined is / are co-administered to a subject in need thereof with chemotherapeutic agent(s) or a second pharmaceutical composition as previously defined

[0052] In other words, the present invention concerns a method for treating a cancer consisting in co-administering to a subject in need thereof (i) extracellular vesicles such as mesenchymal stem cell-derived extracellular vesicles as previously defined or a first pharmaceutical composition as previously defined and (ii) at least one chemotherapeutic agent such as previously defined or a second pharmaceutical composition as previously defined and thereby inhibiting the tumor cell growth and / or proliferation.

[0053] As used herein the term "co-administering" or “co-administration” as used herein means a process whereby the combination of the EVs such as MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition is administered to the same subject. The EVs such as MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition may be administered simultaneously, at essentially the same time, or sequentially.

[0054] The EVs such as MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition need not be administered by means of the same vehicle. The EVs such as MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition may be administered one or more times and the number of administrations of each component of the combination may be the same or different. In addition, the EVs such as the MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition are preferably administered at the same site.

[0055] As used herein, the term "treatment" or "treat" refer to prophylactic, palliative or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting a cancer or suspected to have contracted a cancer as well as subjects who are ill or have been diagnosed as suffering from a cancer, and includes suppression of clinical relapse. The treatment may be administered to a subject having a cancer or who ultimately may acquire a cancer, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a cancer, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

[0056] In some embodiments, the treatment may be administered to a subject whose tumor has been removed by surgery. In particular, in some embodiments, the treatment may be administered to a subject to which cytoreductive surgery has already been performed.

[0057] In some embodiments, the treatment may be administered to a subject whose tumor has not been removed.

[0058] The method of treatment according to the present invention is implemented to treat a subject in need suffering or suspected of suffering from a cancer. In particular, the method of treatment according to the present invention is implemented to treat a subject in need suffering or suspected of suffering from a cancer occurring in a body cavity.

[0059] As used herein, the term “cancer occurring in body cavity” (also known as serosal cancer location) refers to cancer which starts in a body cavity. A body cavity is any space or compartment, or potential space in the body. Body cavity is lined with a layer of cells and is filled with fluid, to protect the organs from damage as the organism moves around. Body cavities form during development, as solid masses of tissue fold inward on themselves, creating pockets in which the organs develop.

[0060] According to the invention, cancers occurring in body cavity include, but are not limited to, head and neck cancer, mesothelioma, pleural metastasis bladder cancer, uterine cancer, pancreatic cancer, esophageal cancer, stomach cancer and peritoneal carcinomatosis.

[0061] In particular, the combination of the EVs such as the MSC-derived EVs and of the chemotherapeutic agent(s) according to the present invention is useful for the treatment of subject suffering from cancer selecting from the list consisting of head and neck cancer, mesothelioma, pleural metastasis, bladder cancer, uterine cancer, pancreatic cancer, esophageal cancer, stomach cancer and peritoneal carcinomatosis. As used herein, the term “head and neck cancer” has its general meaning in the art and refers to cancer in the larynx, throat, mouth, or nose. Head and neck cancers usually begin in the squamous cells that line the moist, mucosal surfaces inside the head and neck (for example, inside the mouth, the nose, and the throat). These squamous cell cancers are often referred to as squamous cell carcinomas of the head and neck. Head and neck cancer include oral cavity cancer (also called mouth cancer), throat cancer such as laryngeal cancer or pharyngeal cancer, nasal and paranasal cancer.

[0062] As used herein, the term “mesothelioma” has its general meaning in the art and refers to a cancer that develops from the thin layer of tissue that covers many of the internal organs (known as the mesothelium). Mesothelioma include pleural mesothelioma and peritoneal mesothelioma.

[0063] As used herein, the term “pleural metastasis” has its general meaning in the art and refers to a cancer that has spread from another organ to the thin membrane (pleura) surrounding the lungs. Pleural metastases are generally associated with metastatic adenocarcinoma and are frequently associated with tumors of the lung, breast, pancreas, and stomach.

[0064] As used herein, the term “bladder cancer” has its general meaning in the art and refers to abnormal growth of the cells of the bladder.

[0065] As used herein, the term “uterine cancer”, also known as womb cancer, has its general meaning in the art and refers to a cancer that develops from the tissues of the uterus. Uterine cancers include endometrial cancer and uterine sarcomas.

[0066] As used herein, the term “pancreatic cancer” has its general meaning in the art and refers to abnormal growth of cells in the pancreas. Pancreatic cancer includes exocrine pancreatic cancer, such as pancreatic ductal adenocarcinoma, and neuroendocrine pancreatic cancer.

[0067] As used herein, the term “stomach cancer”, also known as gastric cancer has its general meaning in the art and refers to an abnormal growth of cells that begins in the stomach. Stomach cancers include gastrointestinal stromal tumors, gastrointestinal carcinoid tumors and gastric carcinoma such as gastric adenocarcinomas. As used herein, the term “peritoneal carcinomatosis” refers to the neoplastic involvement of the peritoneum, typically seen as wide-spread seeding or growth of tumor masses or metastases. Peritoneal carcinomatosis can result from primary or secondary carcinomas.

[0068] Primary peritoneal carcinomas arise from peritoneum cells and since the mesothelium of the peritoneum and the germinal epithelium of the ovary have the same embryologic origin, the peritoneum retains the multipotentiality allowing for the development of a primary carcinoma that can then spread within the peritoneal cavity. Primary carcinomas that cause peritoneal carcinomatosis and are contemplated for treatment using the disclosed methods and agents include malignant mesothelioma, benign papillary mesothelioma, desmoplastic small round cell tumors, peritoneal angiosarcoma, leiomyomatosis peritonealis disseminata (LPD), and peritoneal hemangiomatosis. In addition, ovarian cancer arising in women after bilateral oophorectomy is included as a primary peritoneal cancer that can result in peritoneal carcinomatosis.

[0069] Much more commonly, peritoneal carcinomatosis results from a cancer that arises in an anatonomically separate location and later metastasizes to the peritoneal cavity (secondary peritoneal carcinomas). Numerous cancers can produce peritoneal carcinomatosis including cancers of the endometrium, fallopian tubes, ovaries, uterus, colon, rectum, small bowel, gall bladder, bile duct, appendix, stomach, pancreas, liver and breast.

[0070] In some embodiments, the peritoneal carcinomatosis results from a colorectal cancer. As used herein, the term "colorectal cancer" includes the well- accepted medical definition that defines colorectal cancer as a medical condition characterized by a cancer of cells of the intestinal tract below the small intestine (i.e., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum). Additionally, as used herein, the term "colorectal cancer" also further includes medical conditions, which are characterized by a cancer of cells of the duodenum and small intestine (jejunum and ileum). In some embodiments, the peritoneal carcinomatosis results from an ovarian cancer. As used herein, “ovarian cancer” or “ovarian tumor” includes any tumor, cell mass or micrometastasis derived from, or originating from cells of the ovary. This includes tumors originating from the epithelial cell layer (serous) of the ovary. Ovarian cancer further includes secondary cancers of ovarian origin and further includes recurrent or refractory disease.

[0071] In some embodiments, the peritoneal carcinomatosis is pseudomyxoma peritonei, the peritoneal dissemination of an appendiceal mucinous epithelial neoplasm, a relatively slow growing cancer that is characterized by the excessive production of mucinous ascites. (Smeenk et al, 2007, Cancer Treat. Rev., 33: 138- 145).

[0072] Thus a further object of the present invention relates to a method of treating cancer occurring in body cavity in a subject in need thereof, comprising the steps of co-administering to a subject in need thereof (i) extracellular vesicles such as mesenchymal stem cell-derived extracellular vesicles as previously defined or a first pharmaceutical composition as previously defined and (ii) at least one chemotherapeutic agent such as previously defined or a second pharmaceutical composition as previously defined and thereby inhibiting the tumor cell growth and / or proliferation.

[0073] Thus, the present invention concerns a method for treating a cancer occurring in a body cavity consisting in co-administering to a subject in need thereof (i) a first pharmaceutical composition comprising MSC-derived extracellular vesicles as previously defined and (ii) a second pharmaceutical composition comprising at least one chemotherapeutic agent as previously defined and thereby inhibiting the tumor cell growth and / or proliferation in the subject.

[0074] In particular, a further object of the present invention relates to a method of treating a peritoneal carcinomatosis in a subject in need thereof, comprising the steps of co-administering to a subject in need thereof (i) extracellular vesicles such as mesenchymal stem cell-derived extracellular vesicles as previously defined or a first pharmaceutical composition as previously defined and (ii) at least one chemotherapeutic agent such as previously defined or a second pharmaceutical composition as previously defined and thereby inhibiting the tumor cell growth and / or proliferation.

[0075] In addition, the present invention relates to the combination as previously defined for use in treating a cancer occurring in a body cavity and in particular peritoneal carcinomatosis.

[0076] Thus, the present invention concerns

[0077] - in one hand, chemotherapeutic agent(s) or a second pharmaceutical composition as previously defined for use in treating a cancer occurring in a body cavity and in particular peritoneal carcinomatosis, wherein said chemotherapeutic agent(s) or said second pharmaceutical composition is / are co-administered to a subject in need thereof with extracellular vesicles such as mesenchymal stem cell- derived extracellular vesicles or a first pharmaceutical composition as previously defined and

[0078] - in the other hand, extracellular vesicles such as mesenchymal stem cell- derived extracellular vesicles or a first pharmaceutical composition as previously defined for use in treating a cancer occurring in a body cavity and in particular peritoneal carcinomatosis, wherein said extracellular vesicles such as mesenchymal stem cell-derived extracellular vesicles or said first pharmaceutical composition are co-administered to a subject in need thereof with chemotherapeutic agent(s) or a second pharmaceutical composition as previously defined.

[0079] In the treatment methods according to the present invention, the EVs such as MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition of the combination according to the present invention may be co-administered locally to the area in need of treatment; this can be achieved, for example, by local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.

[0080] In some embodiments, the way to administer the EVs such as MSC-derived EVs or the first pharmaceutical composition and the way to administer the chemotherapeutic agent(s) or the second pharmaceutical composition of the combination according to the present invention may be different.

[0081] In some embodiments, the way to administer the EVs such as MSC-derived EVs or the first pharmaceutical composition and the way to administer the chemotherapeutic agent(s) or the second pharmaceutical composition of the combination according to the present invention may be identical.

[0082] In some embodiments, the EVs such as the MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition of the combination according to the present invention may be co-administered to the area in need of treatment within the subject as an aerosol. In some embodiments, the EVs such as the MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition of the combination according to the present invention can be co-administrated to the subject by spraying them, directly into the abdomen or pleural cavity during a laparoscopy or a thoracoscopy in the form of an aerosol.

[0083] In some embodiments, the EVs such as the MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition of the combination according to the present invention may be co-administered can be co-administrated to the subject by performing pressurized intraperitoneal aerosol chemotherapy (PIPAC) or pressurized intrathoracic aerosol chemotherapy (PITAC) approach.

[0084] As used herein, the expression “pressurized intraperitoneal aerosol chemotherapy (PIPAC)” has its general meaning in the art and refers to a new minimally invasive approach involving spraying drugs, i.e the MSC-derived EVs and the chemotherapeutic agent(s) of the combination according to the present invention, directly into the abdomen of a subject during a laparoscopy in the form of an aerosol. PIPAC approaches are known to those skilled in the art and are briefly described in several articles [Graversen et al, 2018, Pleura Peritoneum, 0128 ; Nadiradze et al, 2020, Cancers, 12(1):34; Tate & Torkington, 2020, BJS Open, 4(2):206-215 ; Alyami et al, 2019, Lancet Oncol., 20(7):e368-e377]. As used herein, the expression “pressurized intrathoracic aerosol chemotherapy (PITAC)” has its general meaning in the art and refers to a new minimally invasive approach involving spraying drugs, i.e the MSC-derived EVs and the chemotherapeutic agent(s) of the combination according to the present invention, directly into the thoracic space of a subject during a thoracoscopy in the form of an aerosol. PITAC approaches are known to those skilled in the art and are briefly described in several articles [Robella et al, 2018, Anticancer Res., 38:929- 932 ; Khosrawipour et al, 2020, Mol. Clin. Oncol., 12(4):350-354 ; Taibi et al, 2020, Ann. Surg. Oncol., 28:3852-3860]. In some embodiments, the EVs such as the MSC-derived EVs or the first pharmaceutical composition and the chemotherapeutic agent(s) or the second pharmaceutical composition of the combination according to the present invention may be repeated at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0085] In some embodiments, when co-administrated with the EVs such as the MSC-derived EVs or the first pharmaceutical composition, the chemotherapeutic agent(s) or the second pharmaceutical composition may be at an administration temperature comprised between 38.5°C and 46.0°C.

[0086] In some embodiments, the co-administration is performed in a subject in need after cytoreductive surgery.

[0087] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0088] FIGURES:

[0089] Figure 1. Calculation of Peritoneal Carcinomatosis Index (PCI) in mice.

[0090] Figure 2. Comparison of PCI obtained for mice into the peritoneal cavity of which CT26 cells have been injected and which have received different treatments. Figure 3. Comparison of PCI obtained for mice into the peritoneal cavity of which MC38 cells have been injected and which have received different treatments.

[0091] Figure 4. Comparison of bioluminescence obtained for mice into the peritoneal cavity of which CT26 cells have been injected and which have received different treatments.

[0092] EXAMPLE:

[0093] Material & Methods

[0094] Extracellular nano vesicles (EVs) derived from mesenchymal stem / stromal cells (MSCs) were produced from a murine (EVm) or a human origin (EVh) according to the method as disclosed in the International application WO 2021 / 099575 Al. When injected in mice, EVm and EVh are in Phostate Buffered Saline (PBS).

[0095] The carcinomatosis model is performed using mice having injection of CT- 26 cells. CT26 is a murin colon cancer cell line. Mice are immunocompetent animals. Intra-peritoneal (ip) injection of CT26 in physiological saline is performed at day 0 using 50 000 cells for one mice. The CT26 cells responded in in vitro study to 2 drugs : Mitomycin and Oxaliplatin diluted in physiological serum.

[0096] Two treatments are performed at day 6 and day 8, using intraperitoneal injection. 9 groups of treatment are performed:

[0097] - a control group « CTRL » with CT26 cells injection and no treatment (shame injection);

[0098] - 2 groups treated with intra-peritoneal chemotherapy :

[0099] - injection Oxaliplatin « OX » : 5 mg / kg in 2 injections,

[0100] - injection of Mitomycin « MITO » : 2 mg / kg in 2 injections ;

[0101] - 2 groups treated with EVm and EVh :

[0102] - injection of EVm « EVm » : 5 x 1010EVm in 2 injections,

[0103] - injection of EVh « EVh » : 5 x 1010EVh in 2 injections ;

[0104] - 4 groups treated with EVs and chemotherapy : - injection Oxaliplatin : 5 mg / kg in 2 injections + EVm 2 injections 5 x IO10« OX +EVm »,

[0105] - injection Oxaliplatin : 5 mg / kg in 2 injections + EVh 2 injections 5 x IO10« OX +EVh »,

[0106] - injection Mitomycine : 2 mg / kg in 2 injections + EVm 2 injections 5 x IO10« MITO + EVm »,

[0107] - injection Mitomycine : 2 mg / kg in 2 injections + EVh 2 injections 5 x IO10« MITO + EVh ».

[0108] At day 9, the bioluminescence is measured in mice after general anaesthesia. Cells fluoresce after intraperitoneal injection of 200 pL of Luciferin. This measurement provides a representation of the intra-peritoneal tumor mass. The results of the bioluminescence analysis have been provided directly by the software after harmonization of the detection scales.

[0109] Final result is done at day 10 using PCI evaluation. PCI is a peritoneal carcinomatosis scoring solution having 0 for no macroscopic carcinomatosis lesion identified to 39 with major carcinomatosis, derived from the human PCI scoring solution. More particularly, to calculate PCI, the abdomen of the mice, after sacrifice at day 10, is divided into 13 regions and, for each region, a score between 1 and 3 is assigned according to the size of the tumors present, score 0 being given to a tumor- free region (Figure 1). The ICP is the sum of the scores for the 13 regions.

[0110] A second cell line had been used using the same protocol : MC38 (a cell line derived from C57BL6 murine colon adenocarcinoma cell). A total of 100 000 cells are injected at day 0 in each mouse, all the rest of the experimental protocol is the same as the one disclosed with CT26 cells.

[0111] Data are represented as mean ± SEM (chi-squared test).

[0112] Results

[0113] In mice injected with CT26 cells

[0114] At day 10, the mean PCI of the groups are respectively at 7.5 / 39 for control group, 3.1 / 39 for OX ip, 2 / 39 for EVh + OX ip, 2.5 / 39 for EVm + OX ip, 2.1 / 39 for MITO ip, 2.4 / 39 for EVh + MITO ip, 0.6 / 39 for EVm + MITO ip, 7.8 / 39 for EVh and 6.6 / 39 for EVm, groups (Figure 2).

[0115] At day 10 the PCI of different groups having CT26 carcinomatosis models are :

[0116] - the PCI is same for control group and EVh and EVm,

[0117] - the PCI is decreased with ip chemotherapy by oxaliplatin or by mitomycin compared with control group,

[0118] - the PCI is decreased with ip chemotherapy by oxaliplatin or by mitomycin and EVm co injections, compared with control group,

[0119] - the PCI is decreased with mitomycin and EVm co-injections, compared with ip chemotherapy by mitomycin.

[0120] In mice injected, with MC38 cells

[0121] At day 10, the mean PCI of the groups are respectively at 9.25 / 39 for control group, 4 / 39 for OX ip, 2.8 / 39 for EVh + OXip , 1.8 / 39 for EVm + OX ip, 3 / 39 for MITO ip, 2 / 39 for EVh + MITO ip, 1.7 / 39 for EVm + MITO ip, 9.8 / 39 for EVh and 6.7 / 39 for EVm, groups (Figure 3).

[0122] At day 10, the PCI of different groups having MC38 carcinomatosis models are :

[0123] - the PCI is same for control group and EVh and EVm,

[0124] - the PCI is decreased with ip chemotherapy by oxaliplatin or by mitomycin compared with control group,

[0125] - the PCI is decreased with ip chemotherapy by oxaliplatin or by mitomycin and EVm co injections, compared with control group,

[0126] - the PCI is decreased with oxaliplatin and EVm co injections, compared with ip chemotherapy by oxaliplatin (p < 0.002).

[0127] Taking together results underline that :

[0128] Using two different models of murin colon carcinomatosis model in immunocompetent animal : - co injection of mitomycin or oxaliplatin drugs using EVm derived from mesenchymal stem / stromal murin cells is highly efficient to decrease the PCI after 2 injections at day 6 and 8 and evaluation at day 10,

[0129] - oxaliplatin and mitomycin ip could be more effective co-injected with EVm.

[0130] In addition, specific experiment had been realized to confirm that human EVs are not affected after a PIP AC procedure using two different PIP AC device : Themasolutions device (Hurrichem) and Skala device (Topol).

[0131] Additional results

[0132] Additional experiments have been carried out measuring bioluminescence in mice injected with CT26 cells and treated with mitomycin ip (Mitomycin group) or with mitomycin and EVm co injections (Mitomycin + EVm group) according to the protocol above explained. The control group has an average bioluminescence of 1160700 ph / s / cm2 / sr. The Mitomycin group has a mean bioluminescence of 922300 ph / s / cm2 / sr. The Mitomycin + EVm group has a mean bioluminescence of 590240 ph / s / cm2 / sr. There is a significant difference between the control group and the Mitomycin + EVm group (p = 0.0008) and between the Mitomycin group and the Mitomycin + EVm group (p = 0.0409) (see Figure 4).

[0133] The results obtained using the measurement of bioluminescence are correlated with the ones obtained with PCI measured for the same mice : correlation according to Pearson is highly positive (r = 0.6536) and reliable (p < 0.0001). Thus, the evaluation of the result with a second bioluminescence technique and the consistency between tumor bioluminescence and measured PCI reinforce the interest of the present invention.

[0134] REFERENCES:

[0135] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. Method for treating a cancer occurring in a body cavity consisting in coadministering to a subject in need thereof (i) a first pharmaceutical composition comprising MSC-derived extracellular vesicles and (ii) a second pharmaceutical composition comprising at least one chemotherapeutic agent and thereby inhibiting the tumor cell growth and / or proliferation in the subject..

2. Method according to claim 1, wherein said at least one chemotherapeutic agent is selected in the group consisting of capecitabine, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, epirubicin, erlotinib, 5 -fluorouracil, gemcitabine, irinotecan, leucovorin, oxaliplatin, paclitaxel, topotecan and any mixture thereof.

3. Method according to claim 1 or 2, wherein said at least one chemotherapeutic agent is selected in the group consisting of cisplatin, doxorubicin, oxaliplatin, CTLA-4 inhibitors such Ipilimumab and Tremelimumab, PD-1 inhibitors such as Cemiplimab, Nivolumab and Pembrolizumab, and PD-L1 inhibitors such as Atezolizumab, Durvalumab and Avelumab and any mixture thereof.

4. Method according to any one of claims 1 to 3, wherein said first pharmaceutical composition contains between 108and 1013MSC-derived extracellular vesicles and particularly between 108and 1012MSC-derived extracellular vesicles / ml of said composition.

5. Method according to any one of claims 1 to 4, wherein said second pharmaceutical composition contains from about 0.01 mg to about 500 mg of said at least one chemotherapeutic agent and, in particular, from 1 mg to about 100 mg of said at least one chemotherapeutic agent / ml of said composition.

6. Method according to any one of claims 1 to 5, wherein said cancer occurring in a body cavity is peritoneal carcinomatosis.

7. Method according to any one of claims 1 to 6, wherein said said first pharmaceutical composition and said second pharmaceutical composition are coadministered to the area in need of treatment within the subject as an aerosol.

8. Method according to any one of claims 1 to 7, wherein said first pharmaceutical composition and said second pharmaceutical composition are coadministered to said subject in need by performing pressurized intraperitoneal aerosol chemotherapy (PIP AC) or pressurized intrathoracic aerosol chemotherapy (PITAC) approach.

9. Method according to any one of claims 1 to 8, wherein, when coadministrated with said first pharmaceutical composition, said second pharmaceutical composition are at an administration temperature comprised between 38.5°C and 46.0°C.

10. Method according to any one of claims 1 to 9, wherein said coadministration is performed in a subject in need after cytoreductive surgery.

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