Non-self multiple stressed cancer cells and their use for vaccination and treatment of cancer - Patents.com

Abstract

The present invention relates to the field of advanced medicines for cell therapy (AMTP).In particular, the present invention relates to a composition comprising stressed HT-29, HCT-116 and LoVo cells, and a composition comprising immunogenic stress proteins produced by these cells in response to stress applied in vitro.The composition allows to simultaneously counter multiple cellular resistance mechanisms observed in situ in cancer cells, and is therefore suitable for vaccination and treatment of cancer in human patients.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the field of advanced medicines for cell therapy (AMTP).In particular, the present invention relates to a composition comprising stressed HT-29, HCT-116 and LoVo cells, and the immunogenic stress proteins produced by these cells in response to stress applied in vitro.This composition allows to simultaneously counter multiple cellular resistance mechanisms observed in cancer cells, and is therefore suitable for vaccination and treatment of cancer in human patients. [Background technology]

[0002] The immune system is based on two defense mechanisms: innate immunity, which is rapid but nonspecific, and adaptive immunity, which is slower but specific and has memory. These two complementary mechanisms confer the ability to fight against external and internal "attacks" either directly, during cell-mediated immune responses, or by recruiting cells through the secretion of active molecules (e.g. immunoglobulins, cytokines, etc.), during humoral immune responses.

[0003] Immunity not only protects the host against the development of primary non-viral cancers, but also shapes tumor immunogenicity. Cancer immunoediting is a process that consists of three stages: elimination (i.e., immune surveillance of cancer), equilibrium, and escape. In the first stage, the immune system fights against tumor growth, which is caused by tumor-associated tissue and environmental changes (recruitment of non-specific cells such as macrophages, NK cells, and DC cells, secretion of anti-proliferative and / or apoptotic molecules, production of cytokines, and CD4 + and CD8 +During the second phase, sensitive cancer cells are eliminated and immune selection of the most resistant cells is activated. The resistance mechanisms activated are resistance to apoptosis, secretion of inhibitory cytokines (TGF-β, IL-10, PGE2 or IDO), changes in antigen presentation (partial or complete loss of expression of class I major histocompatibility complex (MHC)), secretion of neutralizing molecules, and expression of MICA or MICB transcripts to counter the cellular immune system. During this phase, the number of cancer cells destroyed is in equilibrium with the number of resistant cells, hence the name of this phase. The second phase corresponds in particular to the remission period observed during the treatment of cancer patients. During the third phase, cancer cells resistant to the various protective mechanisms of the immune system proliferate. These cancer cells then develop a tumor mass, which is the clinical expression of the physiological escape phenomenon. Related escape phenomena are also observed during the progression and metastasis stages of cancer.

[0004] Anti-cancer treatments include surgery, the purpose of which is primarily to remove or reduce the tumor mass, but which has no actual inhibitory effect on the carcinogenic process, and therefore surgery is usually supplemented with various treatment regimens to completely remove cancer cells.

[0005] Radiation therapy is intended to cause DNA changes in rapidly proliferating cells, such as cancer cells. There are two components to the side effects of radiation therapy: first, not only cancer cells are irradiated, but healthy cells are irradiated as well, which may cause the healthy cells to become "cancerous"; second, cancer cells become resistant to radiation-induced apoptosis by expressing chaperone proteins such as HSPs and GRPs, resulting in an escape phenomenon.

[0006] Chemotherapeutic agents are intended to eliminate cancer cells either by acting on the cancer cells themselves or by inhibiting certain metabolic pathways. Chemotherapeutic agents include agents that directly interact with DNA, such as electrophiles, intercalating agents, or degrading agents, and agents that indirectly interact with DNA, such as DNA synthesis inhibitors (antimetabolites, topoisomerase inhibitors, spindle formation inhibitors, etc.), angiogenesis inhibitors, proteasome inhibitors, etc. Here again, resistance mechanisms are developed by cancer cells, and relapse phenomena with massive tumor growth can be observed despite multi-drug chemotherapy strategies.

[0007] Immunotherapy includes passive immunotherapy, based on the injection of antibodies or cytokines that block receptors, induce cell lysis, stimulate cytotoxicity or reverse the inhibition of apoptosis; and active immunotherapy, also called "immunization by vaccination", in which patients are immunized against cancer with "advanced therapy medicinal products" (ATMPs), a new class of medicinal products defined in Europe by EU Regulation 1394 / 2007.

[0008] Various ATMOs for gene therapy (called GTMPs) are being used in clinical trials for a variety of cancers; to name a few, adenoviral vectors have been used to express p53 in head and neck cancer or CD40L in bladder cancer; oncolytic herpes viruses encoding GM-CSF have been used in melanoma patients; and chimeric antigen receptor (CAR) engineered T cells redirected against various tumor-associated antigens have also been used recently.

[0009] In an attempt to induce clinically meaningful immune responses, AMTPs (called CTMPs) for cell therapy containing autologous dendritic cells (DCs) loaded with tumor antigens have also been used. For example, the FDA and EMA approved a DC-based therapeutic vaccine (sipuleucel-T, trade name "Provenge", developed by Dendreon Pharmaceuticals, LLC) for prostate cancer in 2010 and 2013, respectively, but the European Commission revoked the manufacturing authorization of sipuleucel-T in the European Union in 2015.

[0010] However, AMTP also exhibits a natural evasion phenomenon similar to that described in three-step cancer immunoediting involving the immune system. Thus, there remains a need for therapeutic strategies for the treatment of cancer that provide unique efficacy and / or can contribute to multiple therapeutic strategies.

[0011] Many studies have been conducted to better understand the role of chaperone proteins in the resistance phenomenon of cancer, especially in the resistance to apoptosis.In particular, heat shock proteins (HSPs), glucose-regulated proteins (GRPs) and multidrug resistance proteins (MRPs) are known to be resistance factors and have protective effects.These proteins are produced by cancer cells as a mechanism of resistance as a result of metabolic attacks such as hypoxia, low carbohydrate concentration, physical attacks such as heat stress, irradiation, or chemical attacks such as drugs.

[0012] As a result, trials are being conducted to develop treatment strategies based on the inhibition of these chaperone proteins, but these are not without side effects, including high toxicity levels, with generally mixed results. Trials are also being conducted to use chaperone proteins, particularly HSP70, as agents for immunization against cancer cells in mice. However, this approach focuses on specific chaperone proteins and does not take into account the entire protective mechanism developed by cancer cells.

[0013] To address this problem, the inventors described an in vitro method for obtaining pharmaceutical or vaccine compositions capable of countering cancer cell resistance mechanisms, which is otherwise compatible with the in vitro production of autologous cancer cells with specific resistance mechanisms identical to those developed in situ by cancer cells subjected in vivo to specific stress(es) during the course of a treatment protocol applied to a patient (US Pat. No. 11,096,995; EP Pat. No. 3 057 981). After in vivo administration of these compositions to mice, data showed a favorable effect on tumor volume and weight, confirming the proof of concept.

[0014] Here, the inventors go further and provide novel compositions comprising a selection of non-autologous, multi-stressed cells that simultaneously counteract multidrug resistance mechanisms and are suitable for vaccination of human patients and treatment of various cancers. Summary of the Invention

[0015] The invention is hereinafter particularly as disclosed in the appended claims. In particular, the present invention relates to compositions comprising (i) stressed HT-29, HCT-116 and LoVo cells, and (ii) immunogenic stress proteins produced by these cells in response to stress applied in vitro.

[0016] In one embodiment, stressed HT-29, HCT-116 and LoVo cells have developed resistance mechanisms in response to one or more stresses applied in vitro selected from the group including irradiation, heat stress, chemical stress, metabolic stress and any combination thereof, which result in the production of stress proteins expressed. In one embodiment, stressed HT-29, HCT-116 and LoVo are non-proliferative.

[0017] In one embodiment, the immunogenic stress protein is haptenized. In one embodiment, the immunogenic stress protein is haptenized with any of the following: 2,4-dinitrophenyl (DNP); 2,4-dinitrofluorobenzene; sulfanilic acid; N-iodoacetyl-N'-(5-sulfonic acid-naphthyl)ethylenediamine; aniline; p-aminobenzoic acid; biotin; fluorescein and their derivatives (FITC, TAMRA, and Texas Instruments). Red); digoxigenin; 5-nitro-3-pyrazolecarbamide; 4,5-dimethoxy-2-nitrocinamide; 2-(3,4-dimethoxyphenyl)-quinoline-4-carbamide; 2,1,3-benzoxadiazole-5-carbamide; 3-hydroxy-2-quinoxalinecarbamide; 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL); rotenone isoxazoline; (E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenodi)acetamide; 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid; 2-acetamido-4-methyl-5-thiazolesulfonamide; and p-methoxyphenylpyrazopodophilamide. In one embodiment, the immunogenic stress protein is haptenylated with 2,4-dinitrophenyl (DNP).

[0018] In one embodiment, the composition is a pharmaceutical or vaccine composition and further comprises at least one pharma- ceutically acceptable excipient. In one embodiment, the composition comprises about 10 5 ~about 10 8 The results include HT-29, HCT-116 and LoVo cells subjected to various stresses. The disclosure further relates to this composition for use in treating cancer in a subject in need thereof.The invention also relates to a method of treating cancer in a subject in need thereof comprising administering this composition to the subject.

[0019] The present invention further relates to an intermediate composition comprising (i) one of stressed HT-29 cells, stressed HCT-116 cells and stressed LoVo cells, and (ii) a stress protein, wherein the one of the stressed HT-29 cells, stressed HCT-116 cells or stressed LoVo cells have developed resistance mechanisms in response to in vitro applied (i) metabolic stress, (ii) radiation and (iii) heat stress, which results in the production of the stress protein. The present invention relates to an intermediate composition comprising (i) one of stressed HT-29 cells, stressed HCT-116 cells and stressed LoVo cells, and (ii) a stress protein, wherein the one of the stressed HT-29 cells, stressed HCT-116 cells or stressed LoVo cells have developed a resistance mechanism in response to an in vitro applied (i) metabolic stress, (ii) chemical stress, which results in the production of the stress protein.

[0020] The present invention further provides a method for preparing said intermediate composition, comprising the steps of: a) culturing HT-29, HCT-116 or LoVo cells in a suitable medium; b) subjecting the HT-29, HCT-116 or LoVo cells cultured in step a) to one or more stresses in vitro, wherein said HT29, HCT-116 or LoVo cells develop resistance mechanisms in response to the one or more stresses, thereby producing stress proteins; c) harvesting the stressed HT-29, HCT-116 or LoVo cells together with the stress proteins they produced in step b); d) treating the stressed HT-29, HCT-116 or LoVo cells and the stress proteins they produce, all collected together in step c), with a molecule or method capable of rendering the stress proteins immunogenic. The present invention relates to a method comprising the steps of: In one embodiment, step c) is carried out at least several hours after completion of step b), preferably at least 12 hours or more after completion of step b). In one embodiment, step d) comprises linking or conjugating the stress protein to a means capable of conferring immunogenicity. In one embodiment, the means capable of conferring immunogenicity is a hapten. In one embodiment, the means capable of conferring immunogenicity is 2,4-dinitrophenyl (DNP); 2,4-dinitrofluorobenzene; sulfanilic acid; N-iodoacetyl-N'-(5-sulfonic acid-naphthyl)ethylenediamine; aniline; p-aminobenzoic acid; biotin; fluorescein and derivatives thereof (FITC, TAMRA, and Texas Instruments). Red); digoxigenin; 5-nitro-3-pyrazolecarbamide; 4,5-dimethoxy-2-nitrocinamide; 2-(3,4-dimethoxyphenyl)-quinoline-4-carbamide; 2,1,3-benzoxadiazole-5-carbamide; 3-hydroxy-2-quinoxalinecarbamide; 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL); rotenone isoxazoline; (E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenodi)acetamide; 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid; 2-acetamido-4-methyl-5-thiazolesulfonamide; and p-methoxyphenylpyrazopodophilamide. In one embodiment, the means capable of conferring immunogenicity is 2,4-dinitrophenyl (DNP).

[0021] In one embodiment, the method is for producing an intermediate composition "DS-A" comprising (i) one of stressed HT-29 cells, stressed HCT-116 cells and stressed LoVo cells, and (ii) a stress protein, wherein one of the stressed HT-29 cells, stressed HCT-116 cells or stressed LoVo cells has developed resistance mechanisms in response to in vitro applied (i) metabolic stress, (ii) radiation and (iii) heat stress resulting in the production of a stress protein, and step b) of the method comprises subjecting the HT-29, HCT-116 or LoVo cells cultured in step a) to the following stresses applied in vitro simultaneously or sequentially: (i) in vitro culture in depleted medium, under low oxygen and / or at low pH; preferably in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation with a total dose ranging from about 0.25 to about 25 Gy, preferably from about 1 to about 15 Gy, for a time period preferably ranging from about 1 to about 20 minutes; in vitro irradiation with a total dose preferably of about 10 Gy, for a time period of about 1 to about 5 minutes; and (iii) In vitro heat shock applied at a temperature ranging from about 38° C. to about 45° C. for a time ranging from about 15 minutes to about 4 hours; preferably in vitro heat shock applied at a temperature of about 42° C. for about 60 minutes.

[0022] In one embodiment, the method is for producing an intermediate composition "DS-B" comprising (i) one of stressed HT-29 cells, stressed HCT-116 cells and stressed LoVo cells, and (ii) a stress protein, wherein one of the stressed HT-29 cells, stressed HCT-116 cells or stressed LoVo cells have developed resistance mechanisms in response to in vitro applied (i) metabolic stress and (ii) chemical stress resulting in the production of a stress protein, and step b) of the method comprises subjecting the HT-29, HCT-116 or LoVo cells cultured in step a) to the following stresses applied in vitro simultaneously or sequentially: (i) in vitro culture in depleted medium, under low oxygen and / or at low pH; preferably in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a time period ranging from about 6 hours to about 120 hours. In the latest embodiment, the in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol may be as follows: - when the cell is an HT-29 cell, the in vitro exposure in (ii) is to about 13 μM oxaliplatin for about 72 hours; or - when the cell is a HCT-116 cell, the in vitro exposure in (ii) is to about 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours; or - where the cells are LoVo cells, the in vitro exposure in (ii) is to about 5 μM fluorouracil (5-FU) for about 48 hours.

[0023] The present invention further provides a method for producing the composition of the present invention, comprising the steps of: a. Obtaining six intermediate compositions, the six intermediate compositions comprising: 1) An intermediate composition "DS-A" containing stressed HT-29 cells and stress proteins; 2) an intermediate composition "DS-A" containing stressed HCT-116 cells and stress proteins; 3) an intermediate composition "DS-A" containing stressed LoVo cells and stress proteins; 4) An intermediate composition "DS-B" containing stressed HT-29 cells and stress proteins; 5) an intermediate composition "DS-B" containing stressed HCT-116 cells and stress proteins; 6) an intermediate composition "DS-B" comprising stressed LoVo cells and stress proteins; b. mixing these six intermediate compositions together The present invention relates to a method comprising the steps of: In one embodiment, the six intermediate compositions are mixed together in equal ratios with stressed HT-29, HCT-116 and LoVo cells.

[0024] definition In the present invention, the following terms have the following meanings. As used herein, "HT-29 cells" refers to a human colon adenocarcinoma cell line isolated from a primary tumor of a 44 year old woman in 1964. It contains, among other things, the following sequence variations: - heterozygous for APC p.Blu853Ter (c.2557G>T), -APC p.Thr1556Asnfs * heterozygous for 3(c.4666dupA); -heterozygous for BRAF p.Val600Glu(c.1799T>A), -heterozygous for PIK3CA p.Pro449Thr(c.1345C>A), - homozygous for SMAD4 p.Gln311Ter (c.931C>T), and - Homozygous for TP53 p.Arg273His(c.818G>A).

[0025] This cell line is referenced in the Cellosaurus database under accession number "CVCL_0320" and in the ATCC repository collection under "HTB-38™" and is commercially available from the respective repositories. Other accession numbers for this cell line include "0111" at Banco Celulas do Rio de Janeiro (BCRJ); "ACC299" at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH; "91072201" at General Collection of the European Collection of Authenticated Cell Cultures (ECACC); "30038" at Korean Cell Line Bank (KCLB); and "GDC0149" at China Center for Type Culture Collection (CCTCC). The contents of these collections, in particular the descriptions of the genotype, HLA typing, STR profile and phenotype of this cell line, are incorporated herein by reference.

[0026] As used herein, "HCT-116 cells" refers to a human colon carcinoma cell line isolated from a primary tumor of a 48 year old male in 1981. It contains, among other things, the following sequence variations: -ACVR2A p.Lys437Argfs * homozygous for 5(c.1310delA), -BRCA2 p.Ile2675Aspfs * heterozygous for 6(c.8021dupA); -CDKN2A p.Arg24Serfs * Heterozygous for 20(c.68dupG), -CDKN2A p.Asp74fs * Heterozygous for 21(c.220delG), -CDKN2A p.Glu33Argfs * Heterozygous for 20(c.97delG), - heterozygous for CTNNB1 p.Ser45del (c.133_135delTCT), -EP300 p.Met1470Cysfs * heterozygous for 22(c.4408delA); -Heterozygous for EP300 p.Asn1700Thrfs*9(c.5099delA), -heterozygous for KRAS p.Gly13Asp (c.38G>A), -heterozygous for PIK3CA p.His1047Arg (c.3140A>G), and -TGFBR2 p.Lys128Serfs * Homozygous for 35(c.383delA).

[0027] This cell line is referenced in the Cellosaurus database under accession number "CVCL_0291" and in the ATCC repository collection under "CCL-247™" and is commercially available from the respective repositories. Other accession numbers for this cell line include "0288" at Banco Celulas do Rio de Janeiro (BCRJ); "ACC581" at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH; "91091005" at General Collection of the European Collection of Authenticated Cell Cultures (ECACC); and "10247" at Korean Cell Line Bank (KCLB). The contents of these collections, in particular the descriptions of the genotype, HLA typing, STR profile and phenotype of this cell line, are incorporated herein by reference.

[0028] As used herein, "LoVo cells" refer to a human colorectal adenocarcinoma isolated in 1971 from a fragment of a metastatic tumor in the left supraclavicular region of a 56-year-old male. It contains, among other things, the following sequence variations: -ACVR2A p.Lys437Argfs * heterozygous for 5(c.1310delA), - heterozygous for APC p.Arg1114Ter (c.3340C>T), -APC p.Met1431fs * Heterozygous for 42(c.4289delC), -heterozygous for APC p.Arg2816Gln (c.8447G>A), -B2M p.Leu15Phefs * Heterozygous for 41(c.43_44delCT), -Heterozygous for FBXW7 p.Arg505Cys (c.1513C>T), -heterozygous for KRAS p.Gly13Asp (c.38G>A), - heterozygous for SMAD2 p.Ala292Val (c.875C>T), and -TGFBR2 p.Lys128Serfs * Homozygous for 35(c.383delA).

[0029] This cell line is referenced in the Cellosaurus database under accession number "CVCL_0399" and in the ATCC repository collection under "CCL-229™" and is commercially available from the respective repositories. Other accession numbers for this cell line include "0332" at Banco Celulas do Rio de Janeiro (BCRJ); "ACC350" at Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH; "87060101" at General Collection of the European Collection of Authenticated Cell Cultures (ECACC); and "10229" at Korean Cell Line Bank (KCLB). The contents of these collections, in particular the descriptions of the genotype, HLA typing, STR profile and phenotype of this cell line, are incorporated herein by reference.

[0030] As used herein, "chemotherapeutic agent" refers to any molecule effective in inhibiting tumor growth. Examples of chemotherapeutic agents include those listed in subgroup L01 of the Anatomical Therapeutic Chemistry (ATC) classification system. Further examples of chemotherapeutic agents include, but are not limited to, the following: i. Alkylating agents, such as: Nitrogen mustards, including chlormethine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, chlornaphazine, chlorophosphamide, estramustine, mechlorethamine, mechlorethamine oxide / hydrochloride, novembichin, phenesterine, uracil mustard, etc.; Nitrosoureas, including carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, streptozocin, chlorozotocin, etc.; Alkyl sulfonates, including busulfan, mannosulfan, and treosulfan Aziridines including carboquone, thiotepa, triaziquone, triethylenemelamine, benzodopa, meturedopa, uredopa, etc.; hydrazines including procarbazine, etc.; Triazenes, including dacarbazine, temozolomide, etc.; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaorarnide, trimethylolmelamine, etc.; · and other alkylating agents, including mitobronitol, pipobroman, actinomycin, bleomycin, mitomycin (including mitomycin C, etc.), plicamycin, etc.; ii. Acetogenins, such as bullatacin, bullatacinone, etc.; iii. Benzodiazepines, such as 2-oxoquazepam, 3-hydroxyphenazepam, bromazepam, camazepam, carbrazepam, chlordiazepoxide, cinazepam, cinolazepam, clonazepam, cloniprazepam, clorazepate, ciprazepam, delorazepam, demoxepam, desmethylflunitrazepam, devazepide, diazepam, diclazepam, difludiazepam, doxefazepam, elfazepam, ethyl carfluzepate, ethyl dilazepate, rofuran, Ethyl razepate, Flubromazepam, Fletazepam, Fludiazepam, Flunitrazepam, Flurazepam, Flutemazepam, Flutoprazepam, Fosazepam, Gidazepam, Halazepam, Iclazepam, Irazepine, Kenazepine, Ketazolam, Lorazepam, Lormetazepam, Rufradom, Meclonazepam, Medazepam, Menitrazepam, Metaclazepam, Motrazepam, N-Desalkylflurazepam, Nifoxipam, Nimetazepam, Nitemazepam azepam, nitrazepam, nitrazepate, nordazepam, nortetrazepam, oxazepam, phenazepam, pinazepam, pivoxazepam, prazepam, proflazepam, quazepam, QH-II-66, leclazepam, RO4491533, Ro5-4864, SH-I-048A, surazepam, temazepam, tetrazepam, thifluadm, torfazepam, triflunordazepam, tuclazepam, urdazepam, alfendazam, clozepam Bazam, CP-1414S, lofendazam, triflubazam, gilisopam, GYKI-52466, GYKI-52895, nerisopam, talampanel, tofisopam, adinazolam, alprazolam, bromazolam, clonazolam, estazolam, lualprazolam, flubromazolam, flunitrazolam, nitrazolam, pyrazolam, triazolam, bretazenil, climazolam, EVT-201, FG-8205, flumazenil, GL-II-73, imidazenil, 123I-Iomazenil, L-655,708, Loprazolam, Midazolam, PWZ-029, Remimazolam, Ro15-4513, Ro48-6791, Ro48-8684, Ro4938581, Salmazenil, SH-053-R-CH3-2'F, Cloxazolam, Flutazolam, Haloxazolam, Mexazolam, Oxazolam, Bentazepam, Clothia Zepam, brotizolam, cyclotizolam, deschloroetizolam, etizolam, fluclotizolam, israpafant, JQ1, metizolam, olanzapine, telenzepine, lopirazepam, zapizolam, lazobazam, ripazepam, zolazepam, zomebazam, zometapine, premazepam, clazolam, anthramycin, avizafone, rilmazafone, etc. iv. Antimetabolites, for example: Antifolates, including aminopterin, methotrexate, pemetrexed, pralatrexate, pteropterin, raltitrexed, denopterin, trimetrexate, pemetrexed, etc.; Purine analogues, including pentostatin, cladribine, clofarabine, fludarabine, nelarabine, thioguanine, mercaptopurine, etc.; Pyrimidine analogues, including fluorouracil (5-FU), capecitabine, doxifluridine, tegafur, tegafur / gimeracil / oteracil, carmofur, floxuridine, cytarabine, gemcitabine, azacitidine, decitabine, and others; and Hydroxycarbamide, etc. v. Androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; vi. Antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane, etc.; vii. Folic acid supplements, such as folic acid; viii. Maytansinoids, such as maytansine, ansamitocin, etc.; ix. Platinum analogues, such as platinum, carboplatin, cisplatin, dicycloplatin, nedaplatin, oxaliplatin, satraplatin, etc.; x. Antihormonal agents, such as: Antiestrogens, including tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, toremifene, etc.; Antiandrogens, including flutamide, nilutamide, bicalutamide, leuprolide, goserelin, etc.; xi. Trichothecenes, such as T2 toxin, veracrine A, roridin A, anguidine, etc.; xii. Toxoids, such as cabazitaxel, docetaxel, larotaxel, ortataxel, paclitaxel, tesetaxel, etc.; xiii. Other chemotherapeutic agents, such as camptothecin (and its derivatives: belotecan, cositecan, etirinotecan, pegol, exatecan, gimatecan, irinotecan, raltotecan, rubitecan, siratecan, SN-38 (7-ethyl-10-hydroxycamptothecin), and topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs: adzelesin, carzelesin, and bizelesin); cryptophycin (including cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including its synthetic analogs: KW-2189 and CBI-TMI); eleutherobin, pancratistatin, sarcodictin, spongistatin, aclacinomycin, ausramycin, azaserine, bleomycin, cactinomycin, carabicin, canninomycin, cin;carzinophilin;chromomycin;dactinomycin;daunorubicin;detorubicin;6-diazo-5-oxo-L-norleucine;doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, deoxydoxorubicin, etc.);epirubicin;esorubicin;idarubicin;marcelomycin;mycophenolic acid;nogalanisin nycin);olivomycin;peplomycin;potofilomycin;puromycin;queramycin;rodolubicin;streptomgrin;streptozocin;tubercidin;ubenimex;zinostatin;zorubicin;aceglatone;aldophospharnide glycosides;aminolevulinic acid;amsacrine;bestravcil;bisantrene;edatraxate;defof amine;demecolcine;diaziquone;elfornitine;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidamol;nitracrine;fenamet;pirarubicin;podophyllic acid;2-ethylhydrazide;PSK(registered trademark);razoxane;rhizoxin;sizofiran;spirogennanium;tenuazonic acid;2,2',2''-trichlorotriethylarnine; urethane; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside; 6-thioguanine; vinblastine; etoposide; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; xeloda; ibandronic acid; CPT-11; topoisomerase inhibitor RFS2000; topoisomerase I inhibitor SN38; difluoromethylornithine; retinoic acid; etc.;

[0031] As used herein in the context of a protein, a stress protein, a "detectable level" means that the protein is present in the composition in an amount or concentration that can be detected by means and methods conventionally used by those of skill in the art for the detection of proteins. Such means and methods are well known in the art and include, but are not limited to, mass spectrometry such as liquid chromatography tandem mass spectrometry (LC-MS / MS), as detailed in Examples 3-5 below.

[0032] As used herein, "hapten" refers to a small molecule that elicits an immune response only when conjugated to a larger carrier such as a protein. If a patient generates antibodies against the hapten-carrier conjugate, the hapten may be capable of binding to the antibody but will not normally initiate an immune response; it is only the hapten-carrier conjugate that can perform this function. Examples of haptens include, but are not limited to, 2,4-dinitrophenyl (DNP); 2,4-dinitrofluorobenzene; sulfanilic acid; N-iodoacetyl-N'-(5-sulfonic acid-naphthyl)ethylenediamine; aniline; p-aminobenzoic acid; biotin; fluorescein and their derivatives (FITC, TAMRA, and Texas Instruments). Red); digoxigenin; 5-nitro-3-pyrazolecarbamide; 4,5-dimethoxy-2-nitrocinnamide; 2-(3,4-dimethoxyphenyl)quinoline-4-carbamide; 2,1,3-benzoxadiazole-5-carbamide; 3-hydroxy-2-quinoxalinecarbamide; 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL); rotenone isoxazoline; (E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenodi)acetamide; 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid; 2-acetamido-4-methyl-5-thiazolesulfonamide; and p-methoxyphenylpyrazopodophilamide.

[0033] "Overexpression", and deviations therefrom, as used herein refer to a relative or absolute expression and / or abundance of a marker or protein that is 10% higher, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, in a subject cell or cell population compared to a reference cell or cell population. Alternatively or additionally, overexpression can also refer to a mean fluorescence intensity (MFI) that is at least 10% higher, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, in a subject cell or cell population compared to a reference cell or cell population. In particular, the reference cells are cells (or the reference cell population is a population comprising, preferably consisting of, those cells) from the same cell line as the target cells, but which have not been subjected to the method for producing the composition or intermediate composition described herein; in particular, cells (or the reference cell population is a population comprising, preferably consisting of, those cells) which have been cultured in classical conditions, e.g. in 10% FBS, and have not been subjected to any of the metabolic stresses, irradiation, heat and / or chemical stresses applied in vitro, as described below; or cells (or preferably a population comprising, or preferably consisting of, those cells) from the same cell line as the target cells, which have been cultured in a depleted medium (e.g. a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under hypoxia or at low pH (e.g. below pH 6.5) (i.e. subjected to metabolic stress), and have not been subjected to any of the other stresses, e.g. irradiation, heat and / or chemical stresses applied in vitro, as described below.

[0034] "Underexpression", and deviations therefrom, as used herein refer to a relative or absolute expression and / or abundance of a marker or protein that is 10% lower, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or less, in a subject cell or cell population compared to a reference cell or cell population. Alternatively or additionally, overexpression can also refer to a mean fluorescence intensity (MFI) that is at least 10% lower, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or less, in a subject cell or cell population compared to a reference cell or cell population. In particular, the reference cells are cells (or the reference cell population is a population comprising, preferably consisting of, those cells) from the same cell line as the target cells, but which have not been subjected to the method for producing the composition or intermediate composition described herein; in particular, the reference cells are cells (or the reference cell population is a population comprising, preferably consisting of, those cells) from the same cell line as the target cells, which have been cultured in classical conditions, e.g. in 10% FBS, and have not been subjected to any of the metabolic stresses, irradiation, heat and / or chemical stresses applied in vitro, as described below; or the reference cells are cells (or preferably a population comprising, preferably consisting of, those cells) cultured in a depleted medium (e.g. a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under hypoxia or at low pH (e.g. below pH 6.5) (i.e. subjected to metabolic stress), and have not been subjected to any of the other stresses, e.g. irradiation, heat and / or chemical stresses applied in vitro, as described below.

[0035] As used herein, a "vaccine composition" refers to a composition comprising at least one antigen or immunogen (such as an immunogenic stress and / or resistance protein) and optionally an adjuvant, in a pharma- ceutically acceptable excipient, which is useful for inducing an immune response in a patient upon administration.

[0036] As used herein, "adjuvant" refers to a substance that, upon administration, enhances, augments and / or potentiates the immune response to an antigen (such as, for example, an immunogenic stress and / or resistance protein) in a subject.

[0037] As used herein, "pharmaceutical acceptable excipient" refers to a solid, semi-solid or liquid component of a pharmaceutical or vaccine composition that is not an active ingredient (i.e., neither the stressed HT-29, HCT-116 and LoVo cells nor the immunogenic stress and / or resistance proteins) and that does not produce an adverse, allergic or other untoward reaction when administered to a patient. Most of these pharma- ceutically acceptable excipients are described in detail, for example, in Allen (Ed.), 2017. Ansel's pharmaceutical dosage forms and drug delivery systems (11th ed.). Philadelphia, PA: Wolters Kluwer; Remington, Allen & Adeboye (Eds.), 2013. Remington: The science and practice of pharmacy (22nd ed.). London: Pharmaceutical Press; and Sheskey, Cook & Cable (Eds.), 2017. Handbook of pharmaceutical excipients (8th ed.). London: Pharmaceutical Press. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Drug Formulations (DP / STC-1010) The present invention relates to a composition comprising or consisting of (i) at least one of stressed HT-29, HCT-116, or LoVo cells, and (ii) an immunogenic stress and / or resistance protein, wherein the stress and / or resistance protein is produced by at least one of these HT-29, HCT-116, or LoVo cells in response to a stress applied in vitro.

[0039] In one embodiment, the composition comprises or consists of (i) stressed HT-29 cells and (ii) immunogenic stress and / or resistance proteins produced by these HT-29 cells in response to stress applied in vitro. In one embodiment, the composition comprises or consists of (i) stressed HCT-116 cells and (ii) immunogenic stress and / or resistance proteins produced by these HCT-116 cells in response to stress applied in vitro. In one embodiment, the composition comprises or consists of (i) stressed LoVo cells and (ii) immunogenic stress and / or resistance proteins, which are produced by these LoVo cells in response to stress applied in vitro. In one embodiment, the composition comprises or consists of (i) stressed HT-29 and HCT-116 cells, and (ii) immunogenic stress and / or resistance proteins produced by these HT-29 and HCT-116 cells in response to stress applied in vitro. In one embodiment, the composition comprises or consists of (i) stressed HT-29 and LoVo cells, and (ii) immunogenic stress and / or resistance proteins produced by these HT-29 and LoVo cells in response to stress applied in vitro. In one embodiment, the composition comprises or consists of (i) stressed HCT-116 and LoVo cells, and (ii) immunogenic stress and / or resistance proteins produced by these HCT-116 and LoVo cells in response to stress applied in vitro. In one embodiment, the composition comprises or consists of (i) stressed HT-29, HCT-116 and LoVo cells, and (ii) immunogenic stress and / or resistance proteins produced by these HT-29, HCT-116 and LoVo cells in response to stress applied in vitro.

[0040] Hereinafter, any reference to a composition comprising HT-29, HCT-116 and LoVo cells is intended to encompass a composition comprising one, two or three of HT-29, HCT-116 and LoVo cells. In the present invention, the composition comprises stressed HT-29, HCT-116 and LoVo cells. By "stressed" it is meant that these cells have developed resistance mechanisms in response to stress applied in vitro. As a result, these cells produce stress and / or resistance proteins that form part of the composition.

[0041] In one embodiment, the stress is selected from the group including or consisting of irradiation, thermal stress, chemical stress, metabolic stress, and any combination thereof. In one embodiment, the stresses are combinations and include two, three or more of irradiation, thermal stress, chemical stress and metabolic stress, whether simultaneously or sequentially.

[0042] In one embodiment, the composition of the invention comprises or consists of: stressed HT-29 cells and the stress and / or resistance proteins produced by these HT-29 cells in response to (i) metabolic stress, (ii) radiation and (iii) heat stress applied in vitro; HCT-116 cells subjected to one or more stresses and the stress and / or resistance proteins produced by these HCT-116 cells in response to (i) metabolic stress, (ii) radiation and (iii) heat stress applied in vitro; and LoVo cells subjected to one or more stresses, and the stress and / or resistance proteins produced by these LoVo cells in response to (i) metabolic stress, (ii) radiation and (iii) heat stress applied in vitro.

[0043] In one embodiment, the composition of the invention comprises or consists of: stressed HT-29 cells and the stress and / or resistance proteins produced by these HT-29 cells in response to (i) metabolic stress, and (ii) chemical stress applied in vitro; stressed HCT-116 cells and the stress and / or resistance proteins produced by these HCT-116 cells in response to (i) metabolic stress, and (ii) chemical stress applied in vitro; LoVo cells subjected to one stress and the stress and / or resistance proteins produced by these LoVo cells in response to (i) metabolic stress, and (ii) chemical stress applied in vitro.

[0044] In one embodiment, the composition of the invention comprises or consists of: HT-29 cells subjected to a single stress and the stress and / or resistance proteins produced by these HT-29 cells in response to (i) metabolic stress, (ii) radiation and (iii) heat stress applied in vitro. stressed HCT-116 cells and the stress and / or resistance proteins produced by these HCT-116 cells in response to (i) metabolic stress, (ii) radiation and (iii) heat stress applied in vitro; LoVo cells subjected to one stress and the stress and / or resistance proteins produced by these LoVo cells in response to (i) metabolic stress, and (ii) radiation and (iii) heat stress applied in vitro; stressed HT-29 cells and the stress and / or resistance proteins produced by these HT-29 cells in response to (i) metabolic stress, and (ii) chemical stress applied in vitro; stressed HCT-116 cells and the stress and / or resistance proteins produced by these HCT-116 cells in response to (i) metabolic stress, and (ii) chemical stress applied in vitro; LoVo cells subjected to one stress and the stress and / or resistance proteins produced by these LoVo cells in response to (i) metabolic stress, and (ii) chemical stress applied in vitro.

[0045] In one embodiment, the stress is irradiation. The irradiation is preferably at a dose low enough not to kill or inactivate the cells, but high enough to induce the production of stress and / or resistance proteins. In one embodiment, the irradiation comprises or consists of irradiating the cells with a total dose ranging from about 0.25 to about 25 Gy, preferably about 1 to about 15 Gy, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gy. In one embodiment, the irradiation time is in the range of about 1 to about 20 minutes, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably about 1 to about 5 minutes. In one embodiment, the irradiation comprises or consists of irradiating the cells with a total dose of 10 Gy for a time period of about 1.5 to 2 minutes, i.e., at a dose of about 5 to about 6.6 Gy / min. In one embodiment, the irradiation comprises or consists of irradiating the cells with a total dose of 10 Gy for a time period of about 5 minutes, i.e., at a dose of about 2 Gy / min.

[0046] In one embodiment, the stress is heat stress. The heat stress is preferably at a temperature low enough not to kill or inactivate the cells, but high enough to induce the production of stress and / or resistance proteins. In one embodiment, the heat stress comprises or consists of culturing the cells at a temperature higher than 37°C, preferably at a temperature in the range of about 38°C to about 45°C, such as at a temperature of 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C. In one embodiment, the heat stress is applied to the cells for a time period of about 15 to about 4 hours, preferably for a time period of about 30 minutes to about 2 hours, such as for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes. In one embodiment, the heat stress comprises or consists of culturing the cells at a temperature of about 42° C. for a period of about 60 minutes.

[0047] In one embodiment, the stress is chemical stress. Chemical stress is performed by exposing the cells to at least one or more chemical agents, preferably at a dose low enough not to kill or inactivate the cells, but high enough to induce the production of stress and / or resistance proteins. In one embodiment, the chemical stress comprises or consists of exposing the cells to at least one or more chemotherapeutic agents and / or alcohol. In one embodiment, the chemical stress is applied to the cells for a period ranging from about 6 to about 120 hours, preferably from about 24 to about 96 hours, for example, for 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours. In one embodiment, the chemical stress comprises or consists of exposing the cells, preferably HT-29 cells, to oxaliplatin, preferably at a concentration in the range of about 1 μM to about 20 μM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM, more preferably at a concentration of about 13 μM, for a period of about 60 hours to about 84 hours, preferably for about 72 hours. In one embodiment, the chemical stress comprises or consists of exposing the cells, preferably HCT-116 cells, to SN-38 (7-ethyl-10-hydroxycamptothecin), preferably at a concentration ranging from about 20 nM to about 400 nM, e.g., at a concentration of 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, or 400 nM, more preferably at a concentration of about 31.5 nM, 100 nM or 315 nM, for a period of about 36 hours to about 60 hours, preferably for about 48 hours.In one embodiment, the chemical stress comprises or consists of exposing the cells, preferably LoVo cells, to fluorouracil (5-FU), preferably at a concentration ranging from about 0.5 μM to about 15 μM, e.g., 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15 μM, more preferably at a concentration of about 5 μM, for a period of about 36 hours to about 60 hours, preferably for about 48 hours.

[0048] In one embodiment, the stress is metabolic stress. The metabolic stress is carried out by culturing cells in a non-physiological cell culture medium that does not kill or inactivate cells, but induces the production of stress and / or resistance proteins. In one embodiment, the metabolic stress comprises or consists of culturing cells in a depleted medium (e.g., a medium that partially or completely lacks one or more substances that are useful or even essential for the growth and / or survival of cancer cells), under hypoxia, at low pH (e.g., below pH 6.5). In one embodiment, the metabolic stress comprises or consists of culturing cells in a low serum culture condition, such as, for example, 2% FBS medium (rather than the classically used 10% FBS medium).

[0049] In one embodiment, the composition of the invention comprises or consists of: - stressed HT-29 cells and stress and / or resistance proteins produced by these HT-29 cells in response to (i) in vitro culture under low serum culture conditions in 2% FBS medium, (ii) in vitro irradiation with a total dose of 10 Gy for a time period of about 1 to about 5 minutes, and (iii) in vitro heat shock at a temperature of about 42°C for a period of about 60 minutes; - stressed HCT-116 cells and stress and / or resistance proteins produced by these HCT-116 cells in response to (i) in vitro culture under low serum culture conditions in 2% FBS medium, (ii) in vitro irradiation with a total dose of 10 Gy for a time period of about 1 to about 5 minutes, and (iii) in vitro heat shock at a temperature of about 42°C for a period of about 60 minutes; - stressed LoVo cells and stress and / or resistance proteins produced by these LoVo cells in response to (i) in vitro culture under low serum culture conditions in 2% FBS medium, (ii) in vitro irradiation with a total dose of 10 Gy for a time period of about 1 to about 5 minutes, and (iii) in vitro heat shock at a temperature of about 42°C for about 60 minutes; - stressed HT-29 cells and stress and / or resistance proteins produced by these HT-29 cells in response to (i) in vitro culture under low serum culture conditions in 2% FBS medium, and (ii) in vitro exposure to about 13 μM oxaliplatin for about 72 hours; - stressed HCT-116 cells and stress and / or resistance proteins produced by these HCT-116 cells in response to (i) in vitro culture under low serum culture conditions in 2% FBS medium, and (ii) in vitro exposure to about 31.5 nM, 100 nM or 315 nM of SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours; and -Stressed LoVo cells and stress and / or resistance proteins produced by these LoVo cells in response to (i) in vitro culture under low serum culture conditions in 2% FBS medium, and (ii) in vitro exposure to approximately 5 μM fluorouracil (5-FU) for approximately 48 hours.

[0050] In one embodiment, the composition comprises HT-29, HCT-116 and LoVo cells that are non-proliferative. By "non-proliferative" or "inactive" it is meant that these cells are not capable of undergoing cell proliferation, the process by which a cell grows and divides to produce two daughter cells.

[0051] Those skilled in the art are familiar with the means and methods of rendering cells non-proliferative. For example, cells can be rendered non-proliferative by irradiation at a dose sufficient to kill or inactivate the cells. In one embodiment, irradiation comprises or consists of irradiating the cells with a total dose of 25 Gy or more to render the cells non-proliferative. Alternatively or additionally, cells can be rendered non-proliferative by ethanol fixation, for example with about 10% to about 50% v / v ethanol. Alternatively or additionally, cells can be rendered non-proliferative, for example by at least one freeze-thaw cycle. Alternatively or additionally, cells can be rendered non-proliferative by linking or complexing to a means capable of rendering the cells immunogenic, for example by haptenization.

[0052] Those skilled in the art are also familiar with the means and methods for determining whether cells are non-proliferative or not, for example by performing viability tests by cell culture (to assess complete lack of proliferation) and / or by propidium iodide (to distinguish between live and dead cells).

[0053] In one embodiment, the non-proliferative cells are structurally intact, i.e., they exhibit an intact cell membrane. In one embodiment, the non-proliferative cells are structurally incomplete, i.e., they do not exhibit an intact cell membrane. In the latter case, the cells are present in the composition in the form of membrane fragments, organelles and other cytoplasmic components.

[0054] In one embodiment, the cells become non-proliferative after developing their resistance mechanisms in response to stress applied in vitro, and thus producing stress and / or resistance proteins. In one embodiment, the cells become non-proliferative several hours after stress, preferably more than 12, 24, 36, 48, 60, 72, 84, 96 hours after stress.

[0055] In one embodiment, the composition comprises stressed HT-29, HCT-116 and LoVo cells overexpressing at least one, at least two, at least three, or four of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), CD95 (FAS receptor) and / or CD243 (MDR-1).

[0056] In one embodiment, the composition is compared to HT-29, HCT-116 and / or LoVo cells that have been cultured in classical conditions, e.g., in 10% FBS, and have not been subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress, and (iv) chemical stress; and / or have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but have not been subjected to any of the following in vitro applied (i) irradiation, (ii) heat stress, and (iii) chemical stress. The present invention relates to stressed HT-29, HCT-116 and LoVo cells that express at least one, at least two, at least three, or four of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), CD95 (FAS receptor) and / or CD243 (MDR-1), compared to unstressed HT-29, HCT-116 and / or LoVo cells; at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more. Additionally or alternatively, from HT-29, HCT-116 and / or LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress and (iv) chemical stress; and / or from ...2% FBS, and not subjected to any of the in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress and (iv) chemical stress. At least one, at least two, at least three, or four of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), CD95 (FAS receptor) and / or CD243 (MDR-1) are expressed in the composition; at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, of HT-29, HCT-116 and / or LoVo cell populations that have not been subjected to any of the above-mentioned treatments.Additionally or alternatively, the mean fluorescence intensity (MFI) for at least one, at least two, at least three, or four of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), CD95 (FAS receptor) and / or CD243 (MDR-1) is greater than or equal to 100% for cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress, and (iv) chemical stress. and / or a population of HT-29, HCT-116 and / or LoVo cells that have been subjected to an in vitro applied metabolic stress, e.g., cultured in 2% FBS, but not subjected to any of in vitro applied (i) irradiation, (ii) heat stress and (iii) chemical stress, in the composition at least 2-fold higher, preferably at least 3-fold, 4-fold, 5-fold, 6-fold or more.

[0057] In the present invention, the composition comprises a stress and / or resistance protein. In one embodiment, these stress and / or resistance proteins are associated with the membrane of HT-29, HCT-116 and LoVo cells, i.e. exposed on the surface of HT-29, HCT-116 and LoVo cells, whether on the external or internal surface; and / or contained within HT-29, HCT-116 and LoVo cells, i.e. contained within either their cytoplasm or their organelles; and / or are free in the composition, e.g. because they have been secreted by these cells, i.e. not bound to the membrane of HT-29, HCT-116 and LoVo cells or contained within their cells. In the present invention, the composition comprises a stress and / or resistance protein that is immunogenic. By "immunogenic" or "immunocompetent" is meant that the stress and / or resistance protein is capable of eliciting an immune response in a patient (e.g., the production of antibodies) when administered to said patient.

[0058] In one embodiment, the stress and / or resistance proteins are made immunogenic in the presence of a molecule or by a process capable of rendering them immunogenic, hi one embodiment, the stress and / or resistance proteins are made immunogenic by linkage to or by complex formation with a means capable of conferring immunogenicity. Means and methods capable of conferring immunogenicity are well known to those skilled in the art. In one embodiment, the means capable of conferring immunogenicity comprises or consists of one or more molecules that are not naturally present in HT-29, HCT-116 and LoVo cells or in their environment. Such means are generally referred to as "antigens". Some antigens can induce humoral or cellular immune responses by themselves and are therefore considered to be "immunogens". Some examples of such antigens include, but are not limited to, adjuvants. Those skilled in the art are familiar with adjuvants suitable for use in cancer vaccines, which, when complexed with stress and / or resistance proteins, render them immunogenic. See, for example, Dubensky & Reed, 2010 (Semin Immunol. 22(3):155-61) or Cuzzubbo et al., 2021 (Front Immunol. 11:615240).

[0059] However, some antigens cannot initiate an immune response by themselves and must be prior conjugated to a carrier, e.g., a stress and / or resistance protein. The latter antigens are sometimes referred to as "incomplete antigens" and include, but are not limited to, haptens. Some examples of haptens include, but are not limited to, 2,4-dinitrophenyl (DNP); 2,4-dinitrofluorobenzene; sulfanilic acid; N-iodoacetyl-N'-(5-sulfonic acid-naphthyl)ethylenediamine; aniline; p-aminobenzoic acid; biotin; fluorescein and its derivatives (FITC, TAMRA, and Texas Instruments). Red); digoxigenin; 5-nitro-3-pyrazolecarbamide; 4,5-dimethoxy-2-nitrocinamide; 2-(3,4-dimethoxyphenyl)quinoline-4-carbamide; 2,1,3-benzoxadiazole-5-carbamide; 3-hydroxy-2-quinoxalinecarbamide; 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL); rotenone isoxazoline; (E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenodi)acetamide; 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid; 2-acetamido-4-methyl-5-thiazolesulfonamide; and p-methoxyphenylpyrazopodophilamide.

[0060] In one embodiment, the stress and / or resistance protein is haptenized. In one embodiment, the stress and / or resistance protein is 2,4-dinitrophenylated (DNP). Those skilled in the art are familiar with means and methods for haptenizing proteins, such as stress and / or resistance proteins.

[0061] Another means by which immunogenicity can be conferred involves opsonization, a well-known method that involves the binding of opsonins to stress and / or resistance proteins.

[0062] In one embodiment, the stress and / or resistance protein is selected from the group comprising or consisting of an irradiation resistance protein, a heat stress resistance protein, a chemical stress resistance protein, a metabolic stress resistance protein, and combinations thereof.

[0063] In one embodiment, the composition specifically comprises at least one or more of the following proteins at detectable levels, as compared to either HT-29, HCT-116 or LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress and (iv) chemical stress; and / or as compared to HT-29, HCT-116 and / or LoVo cells that have been subjected to metabolic stress, e.g., by culturing in 2% FBS, but not subjected to any of the following in vitro applied (i) irradiation, (ii) heat stress and (iii) chemical stress; Lipyrimidine tract-binding protein 3, E3 ubiquitin-protein ligase RNF213, serine / arginine-rich splicing factor 8, EH domain-containing protein 4, LIM domain-only protein 7, DNA-dependent RNA polymerase RNA polymerase I subunit RPA2, 2'-5'-oligoadenylate synthase 3, WD repeat and HMG box DNA-binding protein 1, beta-2-glycoprotein 1, serine / threonine protein phosphatase PP1-gamma catalytic subunit, anillin, atypical myosin-Ib, AP-2 complex subunit alpha-2, cyclin-dependent kinase 2, signal transducer and activator of transcription 1-alpha / beta, pumilio homolog 1, ATP-binding cassette subfamily F member 1, RacGTPase-activating protein 1, cingulin, syntaxin-binding protein 3, mitochondrial carnitine / acylcarnitine carrier protein, importin subunit α-7, ribosomal protein S6 kinase α-4, Ras-related protein Rab-5A, ribonucleoside diphosphate reductase large subunit, low molecular weight phosphotyrosine protein phosphatase, ribonucleoside diphosphate reductase subunit M2, ADP-ribosylating factor-like protein 1, dynamin-2, Ras-related protein Rab-13, IST1 homolog, forkhead box protein K1, sorbitol dehydrogenase, Bcl2-like protein 1, tripartite motif-containing protein 29, kinesin-like protein KIF22, methylsterol monooxygenase 1, caveolae-associated protein 1, BRCA1-associated ATM activator 1, protein FA M83H, protein O-mannosyltransferase TMTC3, inhibitor of nuclear transcription factor kappa-B kinase-interacting protein, zinc finger CCCH-type antiviral protein 1, nucleolar protein 9, leucine zipper protein 1, polyhomeotic-like protein 2, tensin-4, LEM domain-containing protein 2, importin-4, Rho guanine nucleotide exchange factor 1, PDZ and LIM domain protein 5, UAP56 interacting factor, MMS19 nucleotide excision repair protein homolog, kinesin-like protein KIF2C, ADP-ribose glycohydrolase MACROD1, kinesin-like protein KIFC1, ATP-dependent RNA helicase DHX33, echinoderm microtubule-associated protein-like 4, Fanconi anemia group I protein, EH domain-containing protein 3, opioid growth factor receptor, gamma-adducin, DNAdC-dU editing enzyme APOBEC-3B, neuroplastin, zinc transporter 1, glutaredoxin-3, cytosolic thymidine kinase, myristoylated alanine-rich C-kinase substrate, UMP-CMP kinase, macrophage capping protein, high mobility group protein HMGI-C, smoothelin, platelet-activating factor acetylhydrolase IB subunit gamma, transcription initiation factor TFIID subunit 9, inactive hydroxysteroid dehydrogenase-like protein 1, borealin, la-associated protein 4, NADH dehydrogenase [ubiquinone]1 alpha subcomplex assembly factor 2, aurora kinase B, Golgi-associated plant pathogenesis-related protein 1, ATP-binding cassette subfamily F member 2, armadillo repeat-containing X-linked protein 3, lagulator complex protein LAMTOR3, putative ATP-dependent RNA helicase DDX20, V-type proton ATPase subunit H, and calcium-binding protein 39.

[0064] In one embodiment, the composition provides a method for the treatment of metabolic disorders in a mammalian animal, comprising administering to said mammalian animal a therapeutic agent, comprising administering to said mammalian animal a therapeutic agent, said therapeutic agent being selected from the group consisting of HT-29, HCT-116 or LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress and (iv) chemical stress; and / or administering to said mammalian animal a therapeutic agent, said therapeutic agent being selected from the group consisting of HT-29, HCT-116 or LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress and (iv) chemical stress; overexpress at least one or more of the following proteins compared to CT-116 and / or LoVo cells: HLA class I histocompatibility complex B alpha chain, HLA class I histocompatibility complex A alpha chain, HLA class I histocompatibility complex C alpha chain, CD9 antigen, nuclear autoantigen Sp-100, ATP-binding cassette subfamily D member 3, ATP-binding cassette subfamily E member 1, ATP-binding cassette subfamily F member 1, ATP-binding cassette subfamily F member 2, Bcl2-like protein 1, Bcl2-binding transcription factor 1, and Bcl2-binding protein 2. factor 1, cytochrome c oxidase subunit 2, mitochondrial cytochrome c oxidase subunit 4 isoform 1, mitochondrial cytochrome c oxidase subunit 5A, peroxisomal acyl-coenzyme A oxidase 1, heat shock protein β1, heat shock protein HSP90β, heat shock cognate 71 kDa protein, heat shock 70 kDa protein 6, heat shock 70 kDa protein 4, ribonuclease inhibitor, inhibitor of nuclear transcription factor kappa-B kinase-interacting protein, Ras-related kinase Protein Rab-5A, Ras-related protein Rab-6A, Ras-related protein Rab-5C, Ras-related protein Rab-7a, Ras-related protein Rab-13, Ras-related protein Rab-25, Ras-related protein Rab-15, Ras-related protein Rab-8A, Ras-related protein Rab-10, Ras-related protein Rab-1b, Ras-related protein Rab-1A, Ras-related protein Rap-1A, Ras-related C3 botulinum toxin substrate 1, Ras-related protein Rab-8B, Ras-related protein Rab-18,Ras-related protein Rab-2c, X-ray repair cross-complementing protein 6, DNA mismatch repair protein Msh6, MMS19 nucleotide excision repair protein homolog, protein transport protein SEc16A, sodium / potassium transporting ATPase subunit α1, facilitative glucose transporter member 1 solute transporter family 2, mitochondrial tricarboxylate transport protein, monocarboxylate transporter member 1, protein transport protein Sec61 subunit β, protein transport protein Sec61 subunit α isoform 1, transport and Golgi organization protein 1 homolog, adenosine 3'-phospho 5'-phosphosulfate transporter 1, transportin-1, and zinc transporter 1.,

[0065] In one embodiment, the composition overexpresses at least one or more of the following membrane proteins: HLA class I histocompatibility antigen B alpha chain, HLA class I histocompatibility antigen A alpha chain, HLA class I histocompatibility antigen B alpha chain, HLA class I histocompatibility antigen C alpha chain, HLA class I histocompatibility antigen D alpha chain, HLA class I histocompatibility antigen E alpha chain, HLA class I histocompatibility antigen F ... Syngene C alpha chain, CD9 antigen, ATP-binding cassette subfamily D member 3, Bcl2-like protein 1, cytochrome c oxidase subunit 2, mitochondrial cytochrome c oxidase subunit 4 isoform 1, inhibitor of nuclear transcription factor kappa B kinase-interacting protein, sodium / potassium transporting ATPase subunit alpha 1, facilitative glucose transporter member 1 solute transporter family 2, mitochondrial tricarboxylate transport protein, monocarboxylate transporter member 1, protein transport protein Sec61 subunit beta, protein transport protein Sec61 subunit alpha isoform 1, transport and Golgi organization protein 1 homolog, adenosine 3'-phospho 5'-phosphosulfate transporter 1, and zinc transporter 1.

[0066] In one embodiment, the composition specifically comprises at least one or more of the following proteins at detectable levels compared to either HT-29, HCT-116 or LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress and (iv) chemical stress; and / or compared to HT-29, HCT-116 and / or LoVo cells that have been subjected to metabolic stress, e.g., by culturing in 2% FBS, but not subjected to any of the following in vitro applied (i) irradiation, (ii) heat stress and (iii) chemical stress: ATP-binding cassette subfamily F member 1, ATP-binding cassette subfamily F member 2, Bcl2-like protein 1, inhibitor of nuclear transcription factor kappa B kinase interacting protein, Ras-related protein Rab-5A, Ras-related protein Rab-13, MMS19 nucleotide excision repair protein homolog, and zinc transporter 1.

[0067] In one embodiment, the composition specifically comprises at least one or more of the following membrane proteins at detectable levels: Bcl2-like protein 1, inhibitor of nuclear transcription factor kappa B kinase interacting protein, and zinc transporter 1, in comparison with either HT-29, HCT-116 or LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, (iii) heat stress and (iv) chemical stress; and / or in comparison with HT-29, HCT-116 and / or LoVo cells that have been subjected to metabolic stress, e.g., by culturing in 2% FBS, but not subjected to any of the following in vitro applied (i) irradiation, (ii) heat stress and (iii) chemical stress.

[0068] In one embodiment, the composition may further comprise a tumor-associated antigen (TAA) and / or a tumor-specific antigen (TSA). "Tumor antigen" refers to an antigenic substance or molecule produced by cancer cells. Tumor antigens are classified into two categories: "tumor-specific antigens" or "TSAs", which are present only on cancer cells and not on non-cancerous cells; and "tumor-associated antigens" or "TAAs", which are present on cancer cells and non-cancerous cells.

[0069] Common TSAs, in particular neoantigens. As used herein, "neoantigen" refers to an abnormal tumor-specific antigen encoded in cancer cells by a gene containing one or more mutations that have arisen, for example, due to genetic instability during carcinogenesis. As a result, the amino acid sequence encoded by this mutated gene may itself contain mutations that result in the production of abnormal proteins not found in normal cells. These mutated proteins are considered non-self or foreign proteins and may therefore be recognized by neoantigen-specific T cell receptors, activating the immune system and triggering an immune system attack against the cancer cells. Neoantigens may also be produced by viral infections, alternative splicing and / or gene rearrangements. See, for example, Zhang et al. (2021. Front Immunol. 12:672356) or Jiang et al. (2019. J Hematol Oncol. 12(1):93).

[0070] In one embodiment, the TAA and / or TSA is specific for HT-29, HCT-116 and LoVo. In one embodiment, the TAAs and / or TSAs are inherently immunogenic. In one embodiment, the TAAs and / or TSAs can be made immunogenic or their immunogenicity can be increased by molecules or methods capable of making them immunogenic. The means by which immunogenicity can be conferred are well known to those skilled in the art and have been described above. These include, in particular, but are not limited to, haptens.

[0071] In one embodiment, these TAAs and / or TSAs are associated with the membrane of HT-29, HCT-116 and LoVo cells, i.e., exposed on the surface of HT-29, HCT-116 and LoVo cells, whether on the external or internal surface; and / or contained within HT-29, HCT-116 and LoVo cells, i.e., contained within either their cytoplasm or their organelles; and / or are free in the composition, e.g., because they have been secreted by these cells, i.e., not associated with the membrane of HT-29, HCT-116 and LoVo cells or contained within their cells.

[0072] In one embodiment, the composition is a pharmaceutical or vaccine composition and further comprises at least one pharma- ceutically acceptable excipient. Pharmaceutically acceptable excipients include, but are not limited to, water, saline, Ringer's solution, dextrose solution and ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate and acetate solutions, gelatin, collagen, Carbopol®, vegetable oils, etc. Also, suitable preservatives, stabilizers, antioxidants, antibacterial agents and buffering agents, such as BHA, BHT, citric acid, ascorbic acid, tetracycline, etc. may be further included. Other examples of pharma- ceutically acceptable excipients that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alum such as aluminum phosphate or aluminum hydroxide, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat. In addition, some pharma- ceutically acceptable excipients include surfactants (e.g., hydroxypropylcellulose), carriers (e.g., solvents and dispersion media, suitable mixtures thereof, including water, ethanol, polyols (e.g., glycerin, propylene glycol, and liquid polyethylene glycol), and vegetable oils, such as peanut oil and sesame oil), isotonic agents (e.g., sugars or sodium chloride), coatings (e.g., lecithin), absorption delaying agents (e.g., aluminum monostearate and gelatin), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal, and the like), buffers (e.g., boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, acetic acid, and the like). sodium, sodium dihydrogen phosphate, etc.), isotonicity agents (e.g., dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, etc.), antioxidants and stabilizers (e.g., sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea, etc.), non-ionic wetting or clarifying agents (e.g., polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol, etc.), viscosity adjusting agents (e.g., dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose, etc.).

[0073] Examples of adjuvants include, but are not limited to, helper peptides, aluminum salts (e.g., aluminum hydroxide (also called alum), aluminum phosphate, etc.), Freund's incomplete adjuvant, Freund's complete adjuvant, saponin, Merck adjuvant 65, SmithKline Beecham adjuvant AS-2, Aquilla adjuvant QS-21, MPL™ immunostimulant, 3d-MPL, LEIF, calcium salts, iron salts, zinc salts, acylated tyrosines, acylated sugars, cationic derivatized polysaccharides, anionic derivatized polysaccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A, muramyl tripeptide phosphatidylethanolamine, cytokines (e.g., interleukin-2, interleukin-12, interleukin-4, interleukin-7, etc.), CpG-containing oligonucleotides, and combinations thereof.

[0074] In one embodiment, the composition comprises about 10 5 ~about 10 8 10 stressed HT-29, HCT-116 and LoVo cells, preferably about 10 6 ~about 10 7 Stressed HT-29, HCT-116 and LoVo cells, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x10 7 In one embodiment, the cells include approximately 3×10 stressed HT-29, HCT-116, and LoVo cells. 6 The results include HT-29, HCT-116 and LoVo cells subjected to various stresses.

[0075] In one embodiment, the composition comprises about 10 6 ~about 10 910 stressed HT-29, HCT-116 and LoVo cells, preferably about 10 per mL of the composition. 6 ~about 10 8 Stressed HT-29, HCT-116 and LoVo cells, for example, about 1x10 per mL of composition 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , or 1x10 8 In one embodiment, the composition contains about 3x10 stressed HT-29, HCT-116 and LoVo cells per mL. 7 The results include HT-29, HCT-116 and LoVo cells subjected to various stresses.

[0076] In one embodiment, the composition comprises equal ratios of stressed HT-29, HCT-116 and LoVo cells (i.e. 1:1:1). In one embodiment, the composition comprises at least 1.5, 2 or 2.5 times more stressed HT-29 cells than stressed HCT-116 or LoVo cells. In one embodiment, the composition comprises at least 1.5, 2 or 2.5 times more stressed HCT-116 cells than stressed HT-29 or LoVo cells. In one embodiment, the composition comprises at least 1.5, 2 or 2.5 times more stressed LoVo cells than stressed HT-29 or HCT-116 cells.

[0077] In one embodiment, the composition comprises: -about 10 5 ~about 10 8 10 stressed HT-29 cells, preferably about 10 6 ~about 10 7 10 stressed HT-29 cells, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x106 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x10 7 HT-29 cells subjected to stress, -about 10 5 ~about 10 8 10 stressed HCT-116 cells, preferably about 10 6 ~about 10 7 10 stressed HCT-116 cells, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x10 7 stressed HCT-116 cells, and -about 10 5 ~about 10 8 10 stressed LoVo cells, preferably about 10 6 ~about 10 7 Stressed LoVo cells, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x10 7 LoVo cells subjected to individual stresses.

[0078] intermediate product The present invention also relates to intermediate compositions useful in the preparation of the above-mentioned compositions. In one embodiment, the intermediate composition comprises at least one pharma- ceutically acceptable excipient as defined above. In one embodiment, the intermediate composition comprises or consists of (i) stressed HT-29, HCT-116 and LoVo cells, and (ii) stress and / or resistance proteins produced by these HT-29, HCT-116 and LoVo cells in response to in vitro applied (i) metabolic stress, (ii) irradiation and (iii) heat stress. Radiation, heat stress and metabolic stress have been described previously. In one embodiment, the stress and / or resistance protein is immunogenic. Means and methods for making the stress and / or resistance protein immunogenic have been described previously.

[0079] In one embodiment, the intermediate composition may further comprise a tumor-associated antigen (TAA) and / or a tumor-specific antigen (TSA), as described above. In one embodiment, the TAA and / or TSA are specific for HT-29, HCT-116 and LoVo cells. In one embodiment, the TAA and / or TSA are intrinsically immunogenic. In one embodiment, the TAA and / or TSA may be made immunogenic or their immunogenicity may be increased by linking or complexing with a means capable of conferring immunogenicity. Means capable of conferring immunogenicity are well known to those skilled in the art and have been described above. These include, but are not limited to, haptens, among others.

[0080] In one embodiment, the intermediate composition comprises or consists of (i) stressed HT-29 cells, and (ii) stress and / or resistance proteins produced by these HT-29 cells in response to: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) irradiation with a total dose ranging from about 0.25 to about 25 Gy, preferably about 1 to about 15 Gy, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gy (in one embodiment, the irradiation time is in the range of about 1 to about 20 minutes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably in the range of about 1 to about 5 minutes); and (iii) In vitro heat shock at a temperature in the range of about 38° C. to about 45° C., e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., applied to the cells for a period of about 15 to about 4 hours, preferably about 30 minutes to about 2 hours, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

[0081] In one embodiment, the intermediate composition comprises or consists of (i) stressed HT-29 cells, and (ii) stress and / or resistance proteins produced by these HT-29 cells in response to: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation for about 1 to about 5 minutes with a total dose of 10 Gy; and (iii) In vitro heat shock at a temperature of about 42° C. for about 60 minutes. This intermediate composition is referred to herein as "HT-29 DS-A."

[0082] In one embodiment, the HT-29 DS-A comprises a stress and / or resistance protein selected from the group including or consisting of an irradiation resistance protein, a heat stress resistance protein, a metabolic stress resistance protein, and combinations thereof. In one embodiment, the HT-29 DS-A cells overexpress at least one, at least two, or three of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1) and / or CD107 (LAMP-1). In one embodiment, HT-29 DS-A cells express at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of at least one, at least two, or three of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), and / or CD107 (LAMP-1), compared to HT-29 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of (i) metabolic stress, (ii) irradiation, and (iii) heat stress applied in vitro; and / or compared to HT-29 cells that have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but not subjected to any of (i) irradiation, and (ii) heat stress applied in vitro. Additionally or alternatively, at least one, at least two, or three of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), and / or CD107 (LAMP-1) are expressed at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, in a population of HT-29 DS-A cells than a population of HT-29 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of (i) metabolic stress, (ii) irradiation, and (iii) heat stress applied in vitro; and / or than a population of HT-29 cells that have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but not subjected to any of (i) radiation, and (ii) heat stress applied in vitro.Additionally or alternatively, the mean fluorescence intensity (MFI) of at least one, at least two, or three of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1) and / or CD107 (LAMP-1) is at least 2-fold higher, preferably at least 3-fold, 4-fold, 5-fold, 6-fold or higher, in a population of HT-29 DS-A cells than a population of HT-29 cells cultured in classical conditions, e.g. in 10% FBS, and not subjected to any of in vitro applied (i) metabolic stress, (ii) irradiation, and (iii) heat stress; and / or than a population of HT-29 cells that have been subjected to an in vitro applied metabolic stress, e.g. cultured in 2% FBS, but not subjected to any of in vitro applied (i) radiation, and (ii) heat stress.

[0083] In one embodiment, compared to HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, HT-29 DS-A cells overexpress at least one or more of the following proteins: poly(rC) binding protein 2, rho guanine nucleotide exchange factor 1, golgin subfamily B member 1, beta-2-glycoprotein 1, plakophilin-3, protein FAM83H, protein transport protein Sec16A, reverse formin-2, anilene, protein ECT2, plectin, epiplakin, proliferation marker protein Ki-67, endoplasmic reticulum integral membrane protein VIP36, and lysosomal membrane glycoprotein 1.

[0084] In one embodiment, compared to HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, HT-29 DS-A cells differentially express at detectable levels at least one or more of the following proteins: poly(rC) binding protein 2, rho guanine nucleotide exchange factor 1, golgin subfamily B member 1, beta-2-glycoprotein 1, plakophilin-3, protein FAM83H, protein transport protein Sec16A, reverse formin-2, anillin, and protein ECT2.

[0085] In one embodiment, compared to HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, HT-29 DS-A cells overexpress at least one or more of the following membrane and / or cell surface proteins: endoplasmic reticulum integral membrane protein VIP36, lysosomal membrane glycoprotein 1, ribosome-associated protein 1, Kunitz-type protease inhibitor 2, lysophospholipid acyltransferase 5, emerin, protein LYRIC, very long chain fatty acid elongase protein 1, and protein transport protein Sec61 subunit γ.

[0086] In one embodiment, the HT-29 cells are subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to either (i) radiation or (ii) heat stress applied in vitro. DS-A cells express the following cell surface proteins: heat shock-associated 70 kDa protein, annexin, anoctamin-6, immunoglobulin superfamily member 3, serotransferrin, tumor necrosis factor receptor superfamily member 10B, clusterin, furin, HLA class II histocompatibility complex gamma chain, CD109 antigen, chloride intracellular channel protein 4, protocadherin lipid 1, natural resistance-associated macrophage protein 2, tumor necrosis factor receptor superfamily member 10A, calpain-5, MHC class I polypeptide-related sequence A, high mobility group protein B1, tetraspanin-15, UL16-binding protein 2, integrin beta-7, sonic hedgehog protein, toll-like receptor 3, beta-2-glycoprotein 1, tissue factor, proprotein convertase subtilisin / kexin type 6, endothelial protein C receptor, volume-sensitive anion channel subunit LRRC8A, and cadherin EGF The cells overexpress at least one or more of the following: LAG seven-transmembrane G-type receptor 3, zinc transporter ZIP6, HLA class II histocompatibility complex DM alpha chain, cystine / glutamate transporter, lysophosphatidic acid receptor 2, syndecan-1, hyaluronidase-2, integrin α4, histidine-rich glycoprotein, transforming growth factor beta-1 proprotein, and metalloproteinase inhibitor 2.

[0087] In one embodiment, the intermediate composition comprises or consists of (i) stressed HCT-116 cells, and (ii) stress and / or resistance proteins produced by these HCT-116 cells in response to: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) irradiation with a total dose ranging from about 0.25 to about 25 Gy, preferably from about 1 to about 15 Gy, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gy (in one embodiment, the irradiation time is in the range of about 1 to about 20 minutes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably in the range of about 1 to about 5 minutes); and (iii) In vitro heat shock at a temperature in the range of about 38° C. to about 45° C., e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., applied to the cells for a period of about 15 to about 4 hours, preferably about 30 minutes to about 2 hours, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

[0088] In one embodiment, the intermediate composition comprises or consists of (i) stressed HCT-116 cells, and (ii) stress and / or resistance proteins produced by these HCT-116 cells in response to: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation for about 1 to about 5 minutes with a total dose of 10 Gy; and (iii) In vitro heat shock at a temperature of about 42° C. for about 60 minutes. This intermediate composition is referred to herein as "HCT-116 DS-A."

[0089] In one embodiment, the HCT-116 DS-A comprises a stress and / or resistance protein selected from the group including or consisting of an irradiation resistance protein, a heat stress resistance protein, a metabolic stress resistance protein, and combinations thereof. In one embodiment, the HCT-116 DS-A cells overexpress at least one or two of the following markers: Cmhsp70.1 (HSP70) and / or CD227 (MUC1).

[0090] In one embodiment, HCT-116 DS-A cells express at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of at least one or two of the following markers: Cmhsp70.1 (HSP70), and / or CD227 (MUC1), compared to HCT-116 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, and (iii) heat stress; and / or compared to HCT-116 cells that have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but not subjected to any of the following in vitro applied (i) irradiation, and (ii) heat stress; Additionally or alternatively, at least one or two of the following markers: Cmhsp70.1 (HSP70) and CD227 (MUC1) are expressed by at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more in a population of HCT-116 DS-A cells than a population of HCT-116 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of (i) metabolic stress, (ii) irradiation, and (iii) heat stress applied in vitro; and / or than a population of HCT-116 cells that have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but not subjected to any of (i) irradiation, and (ii) heat stress applied in vitro.Additionally or alternatively, the mean fluorescence intensity (MFI) of at least one or two of the following markers: Cmhsp70.1 (HSP70) and CD227 (MUC1) is at least 2-fold higher, preferably at least 3-fold, 4-fold, 5-fold, 6-fold or more higher, in a population of HCT-116 DS-A cells than a population of HCT-116 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of in vitro applied (i) metabolic stress, (ii) irradiation, and (iii) heat stress; and / or than a population of HCT-116 cells that have been subjected to an in vitro applied metabolic stress, e.g., cultured in 2% FBS, but not subjected to any of in vitro applied (i) radiation, and (ii) heat stress.

[0091] In one embodiment, compared to HCT-116 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, HCT-116 DS-A cells overexpress at least one or more of the following proteins: poly(rC) binding protein 2, rho guanine nucleotide exchange factor 1, golgin subfamily B member 1, beta-2-glycoprotein 1, plakophilin-3, protein FAM83H, protein transport protein Sec16A, reverse formin-2, anilene, protein ECT2, plectin, epiplakin, proliferation marker protein Ki-67, endoplasmic reticulum integral membrane protein VIP36, and lysosomal membrane glycoprotein 1.

[0092] In one embodiment, compared to HCT-116 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, HCT-116 DS-A cells differentially express at least one or more of the following proteins at detectable levels: poly(rC) binding protein 2, rho guanine nucleotide exchange factor 1, golgin subfamily B member 1, beta-2-glycoprotein 1, plakophilin-3, protein FAM83H, protein transport protein Sec16A, reverse formin-2, anillin, and protein ECT2.

[0093] In one embodiment, compared to HCT-116 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to any of (i) radiation and (ii) heat stress applied in vitro, HCT-116 DS-A cells overexpress at least one or more of the following membrane and / or cell surface proteins: integrin α6, lysosomal membrane glycoprotein lysosomal membrane glycoprotein 1, ribosome-associated binding protein 1, delta(14) sterol reductase LBR, mitochondrial proton / calcium exchanger protein, RRP12-like protein, extended synaptotagmin (ES)-dependent endothelial cell death protein (EGFR), ... synaptotagmin-1, protein transport protein Sec61 subunit alpha isoform 1, solute transporter family 2, facilitative glucose transporter member 1, desmoglein-2, kinectin, protein LYRIC, sphingosine-1-phosphate lyase 1, vesicle-associated membrane protein-associated protein A, lysophospholipid acyltransferase 7, signal recognition particle receptor subunit beta, torsin-1A-interacting protein 1, ER membrane protein complex subunit 1, sterol-4α-carboxylate-3-dehydrogenase (decarboxylation) , membrane-associated progesterone receptor component 1, CD44 antigen MICOS complex subunit MIC26, transmembrane emp24 domain-containing protein 4, ORM1-like protein 2, dolichol phosphate mannosyltransferase subunit 3, cytoskeleton-associated protein 4, transmembrane protein 43, retinol dehydrogenase 11, signal peptidase complex subunit 2, serine palmitoyltransferase 2, coiled-coil domain-containing protein 47, very long-chain enoyl-CoA reductase, protocadherin Fat1, transmembrane emp24 domain-containing protein 7, caveolin-1, NF-X1 type zinc finger protein NFXL1, receptor expression enhancing protein 6, receptor expression promoting protein 6, mitochondrial HIG1 domain family member 2A, small integral membrane protein 20, DH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13, DH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3, mitochondrial thiamine pyrophosphate carrier, mitochondrial fission 1 protein, amine oxidase [flavin-containing] B, mitochondrial inner membrane protein membrane protein OXA1L, 2-hydroxyacyl-CoA lyase 2, nicalin, plasma membrane calcium transporting ATPase 1, myoferlin, and cation-independent mannose 6-phosphate receptor.

[0094] In one embodiment, the intermediate composition comprises or consists of (i) stressed LoVo cells, and (ii) stress and / or resistance proteins produced by these LoVo cells in response to: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) irradiation with a total dose ranging from about 0.25 to about 25 Gy, preferably about 1 to about 15 Gy, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gy (in one embodiment, the irradiation time is in the range of about 1 to about 20 minutes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably in the range of about 1 to about 5 minutes); and (iii) In vitro heat shock at a temperature in the range of about 38° C. to about 45° C., e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., applied to the cells for a period of about 15 to about 4 hours, preferably about 30 minutes to about 2 hours, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

[0095] In one embodiment, the intermediate composition comprises or consists of (i) stressed LoVo cells, and (ii) stress and / or resistance proteins produced by these LoVo cells in response to: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation for about 1 to about 5 minutes with a total dose of 10 Gy; and (iii) In vitro heat shock at a temperature of about 42° C. for about 60 minutes. This intermediate composition is referred to herein as "LoVo DS-A."

[0096] In one embodiment, the LoVo DS-A comprises a stress and / or resistance protein selected from the group including or consisting of a radiation resistance protein, a heat stress resistance protein, a metabolic stress resistance protein, and combinations thereof.

[0097] In one embodiment, LoVo DS-A cells overexpress at least one, at least two, or three of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1) and / or CD107 (LAMP-1).

[0098] In one embodiment, LoVo DS-A cells express at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), and / or CD107 (LAMP-1), compared to LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to any of the following in vitro applied (i) metabolic stress, (ii) irradiation, and (iii) heat stress; and / or compared to LoVo cells that have been subjected to metabolic stress, e.g., by culturing in 2% FBS, but not subjected to any of the following in vitro applied (i) irradiation, and (ii) heat stress. Additionally or alternatively, at least one, at least two, or three of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1), and / or CD107 (LAMP-1) are expressed by at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more in a population of LoVo DS-A cells than a population of LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) irradiation, and (iii) heat stress; and / or than a population of LoVo cells that have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but not subjected to in vitro applied (i) radiation, and (ii) heat stress.Additionally or alternatively, the mean fluorescence intensity (MFI) of at least one, at least two or three of the following markers: Cmhsp70.1 (HSP70), CD227 (MUC1) and CD107 (LAMP-1) is at least 2-fold higher, preferably at least 3-fold, 4-fold, 5-fold, 6-fold or higher in a population of LoVo DS-A cells than a population of LoVo cells cultured in classical conditions, e.g. in 10% FBS, and not subjected to any of in vitro applied (i) metabolic stress, (ii) irradiation, and (iii) heat stress; and / or than a population of LoVo cells that have been subjected to in vitro applied metabolic stress, e.g. cultured in 2% FBS, but not subjected to any of in vitro applied (i) radiation, and (ii) heat stress.

[0099] In one embodiment, compared to LoVo cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, LoVo DS-A cells overexpress at least one or more of the following proteins: poly(rC) binding protein 2, rho guanine nucleotide exchange factor 1, golgin subfamily B member 1, beta-2-glycoprotein 1, plakophilin-3, protein FAM83H, protein transport protein Sec16A, reverse formin-2, anilene, protein ECT2, plectin, epiplakin, proliferation marker protein Ki-67, endoplasmic reticulum integral membrane protein VIP36, and lysosomal membrane glycoprotein 1.

[0100] In one embodiment, compared to LoVo cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, LoVo DS-A cells differentially express at least one or more of the following proteins at detectable levels: poly(rC) binding protein 2, rho guanine nucleotide exchange factor 1, golgin subfamily B member 1, beta-2-glycoprotein 1, plakophilin-3, protein FAM83H, protein transport protein Sec16A, reverse formin-2, anillin, and protein ECT2.

[0101] In one embodiment, compared to LoVo cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied (i) radiation and (ii) heat stress, LoVo DS-A cells overexpress at least one or more of the following membrane and / or cell surface proteins: endoplasmic reticulum integral membrane protein VIP36, nucleolar complex protein 4 homolog, signal peptidase complex catalytic subunit SEC11A, signal recognition particle receptor subunit β, microsomal glutathione S-transferase 1, DPH-cytochrome P450 reductase, very long chain (3R)-3-hydroxyacyl-CoA dehydratase 3, mannosyl oligosaccharide glucosidase, oxysterol binding protein-associated protein 8, receptor expression-enhancing protein 5, MICOS complex subunit MIC26, β-1,3-galactosyl-O-glycosyl glycoprotein β-1,6-N-acetylglucosaminyltransferase 3, mitochondrial ATP synthase membrane subunit DAPIT, mitochondrial ATP synthase subunit f, CDGSH iron-sulfur domain-containing protein 2, vitamin K epoxide reductase complex subunit 1-like protein 1, protein FAM162A, erlin-1, long-chain fatty acid-CoA ligase 1, CYB5B_human cytochrome b5 type B, retinol dehydrogenase 11, transmembrane emp24 domain-containing protein 2, very long-chain enoyl-CoA reductase, 3-β-hydroxysteroid-dehydrogenase ruta(8), delta(7)-isomerase, monocarboxylate transporter 1, HLA class I histocompatibility complex B alpha chain, erlin-2, CD9 antigen, ATP synthase protein 8, mitochondrial fission process protein 1, cytochrome c oxidase subunit NDUFA4, DH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13, DH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 4, DH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3, mitochondrial fission 1 protein, and BRI3 binding protein.

[0102] In one embodiment, the intermediate composition comprises or consists of (i) stressed HT-29, HCT-116 and LoVo cells, and (ii) stress and / or resistance proteins produced by these HT-29, HCT-116 and LoVo cells in response to in vitro applied (i) metabolic stress, and (ii) chemical stress. Chemical and metabolic stresses have been described previously. In one embodiment, the stress and / or resistance protein is immunogenic. Means and methods for making the stress and / or resistance protein immunogenic have been described above.

[0103] In one embodiment, the intermediate composition may further comprise a tumor-associated antigen (TAA) and / or a tumor-specific antigen (TSA), as described above. In one embodiment, the TAA and / or TSA are specific for HT-29, HCT-116 and LoVo cells. In one embodiment, the TAA and / or TSA are intrinsically immunogenic. In one embodiment, the TAA and / or TSA may be made immunogenic or their immunogenicity may be increased by linking or complexing with a means capable of conferring immunogenicity. Means capable of conferring immunogenicity are well known to those skilled in the art and have been described above. These include, but are not limited to, haptens, among others.

[0104] In one embodiment, the intermediate composition comprises or consists of (i) stressed HT-29 cells, and (ii) stress and / or resistance proteins produced by these HT-29 cells in response to: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a period ranging from about 6 hours to about 120 hours, preferably from about 24 hours to about 96 hours, e.g., 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours.

[0105] In one embodiment, the intermediate composition comprises or consists of (i) stressed HT-29 cells, and (ii) stress and / or resistance proteins produced by these HT-29 cells in response to: (i) in vitro culture in low serum culture conditions in 2% FBS medium, and (ii) In vitro exposure to about 13 μM oxaliplatin for about 72 hours. This intermediate composition is referred to herein as "HT-29 DS-B."

[0106] In one embodiment, the HT-29 DS-B comprises a stress and / or resistance protein selected from the group including or consisting of an irradiation resistance protein, a heat stress resistance protein, a metabolic stress resistance protein, and combinations thereof.

[0107] In one embodiment, HT-29 DS-B cells overexpress at least one, at least two or three of the following markers: CD54 (ICAM-1), CD95 (FAS receptor) and / or CD107 (LAMP-1).

[0108] In one embodiment, HT-29 DS-B cells express at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of at least one, at least two or three of the following markers: CD54 (ICAM-1), CD95 (FAS receptor), and / or CD107 (LAMP-1), compared to HT-29 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) chemical stress (e.g., in vitro exposure to about 13 μM oxaliplatin for about 72 hours); and / or compared to HT-29 cells that have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but not subjected to in vitro applied chemical stress (e.g., in vitro exposure to about 13 μM oxaliplatin for about 72 hours). Additionally or alternatively, at least one, at least two or three of the following markers: CD54 (ICAM-1), CD95 (FAS receptor), and / or CD107 (LAMP-1) are expressed at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, in a population of HT-29 DS-B cells than a population of HT-29 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) chemical stress (e.g., in vitro exposure to about 13 μM oxaliplatin for about 72 hours); and / or than a population of HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied chemical stress (e.g., in vitro exposure to about 13 μM oxaliplatin for about 72 hours).Additionally or alternatively, the mean fluorescence intensity (MFI) of at least one, at least two or three of the following markers: CD54 (ICAM-1), CD95 (FAS receptor), and / or CD107 (LAMP-1) is at least 2-fold higher, preferably at least 3-fold, 4-fold, 5-fold, 6-fold or higher, in a population of HT-29 DS-B cells than a population of HT-29 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) chemical stress (e.g., in vitro exposure to about 13 μM oxaliplatin for about 72 hours); and / or than a population of HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied chemical stress (e.g., in vitro exposure to about 13 μM oxaliplatin for about 72 hours).

[0109] In one embodiment, compared to HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro, HT-29 DS-B cells overexpress at least one or more of the following proteins: beta-2-glycoprotein 1, histone H2A type 1, poly(rC)-binding protein 2, kinectin, plectin, and NADH dehydrogenase [ubiquinone]1 alpha subcomplex subunit 7.

[0110] In one embodiment, HT-29 DS-B cells differentially express at least one or more of the following proteins at detectable levels: beta-2-glycoprotein 1, histone H2A type 1, and poly(rC) binding protein 2, as compared to HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro.

[0111] In one embodiment, HT-29 DS-B cells overexpress at least one or more of the following membrane and / or cell surface proteins: ribosome-associated protein 1, and NADPH-cytochrome P450 reductase, as compared to HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro.

[0112] In one embodiment, compared to HT-29 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro, HT-29 DS-B cells overexpress at least one or more of the following cell surface proteins: CD109 antigen, HLA class II histocompatibility complex gamma chain, HLA class I histocompatibility complex alpha chain F, hyaluronan-mediated motility receptor, integrin beta8, integrin beta3, proprotein convertase subtilisin / kexin type 6, clusterin, serotransferrin, natural resistance-associated macrophage protein 2, MHC class I polypeptide-related sequence A, tumor necrosis factor receptor superfamily member 10B, endothelial protein C receptor, cadherin EGF. LAG seven-transmembrane G-type receptor 3, tumor necrosis factor receptor superfamily member 10A, cystine / glutamate transporter, tissue factor, transforming growth factor beta-1 proprotein, immunoglobulin superfamily member 3, anoctamin-6, metalloproteinase inhibitor 2, toll-like receptor 3, volume-regulated anion channel subunit LRRC8A, tetraspanin-15, zinc transporter ZIP6, furin, protocadherin fat 1, hyaluronidase-2, lysophosphatidic acid receptor 2, high-mobility group protein B1, chloride intracellular channel protein 4, UL16 binding protein 2, calpain-5, annexin A9, histidine-rich glycoprotein, integrin α4, and heat shock-related 70 kDa protein 2.

[0113] In one embodiment, the intermediate composition comprises or consists of (i) stressed HCT-116 cells and (ii) stress and / or resistance proteins produced by these HCT-116 cells in response to: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a period ranging from about 6 hours to about 120 hours, preferably from about 24 hours to about 96 hours, e.g., 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours.

[0114] In one embodiment, the intermediate composition comprises or consists of (i) stressed HCT-116 cells, and (ii) stress and / or resistance proteins produced by these HCT-116 cells in response to: (i) in vitro culture in low serum culture conditions in 2% FBS medium, and (ii) In vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours. This intermediate composition is referred to herein as "HCT-116 DS-B."

[0115] In one embodiment, the HCT-116 DS-B comprises a stress and / or resistance protein selected from the group including or consisting of a radiation resistance protein, a heat stress resistance protein, a metabolic stress resistance protein, and combinations thereof.

[0116] In one embodiment, HCT-116 DS-B cells overexpress the following markers: CD66 (CEA).

[0117] In one embodiment, HCT-116 DS-B cells exhibit a significantly increased metabolic activity in comparison to HCT-116 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) chemical stress (e.g., in vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours); and / or subjected to metabolic stress, e.g., by being cultured in 2% FBS. In one embodiment, the HCT-116 cells express at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, of the marker CD66 (CEA) as compared to HCT-116 cells that have been subjected to in vitro applied chemical stress (e.g., in vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours). Additionally or alternatively, the marker CD66 (CEA) is increased or decreased from a population of HCT-116 cells that have been cultured in classical conditions, e.g., in 10% FBS, and that have not been subjected to either (i) metabolic stress, (ii) chemical stress applied in vitro (e.g., in vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours); and / or from a population of HCT-116 cells that have been subjected to metabolic stress, e.g., by being cultured in 2% FBS, but not subjected to chemical stress applied in vitro (e.g., in vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours). It is expressed in at least 10% of the population of DS-B cells, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.Additionally or alternatively, the mean fluorescence intensity (MFI) of the marker CD66 (CEA) is significantly higher than that of HCT-116 cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to either (i) metabolic stress, (ii) chemical stress applied in vitro (e.g., in vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours), and / or than that of HCT-116 cells cultured in 2% FBS, and not subjected to chemical stress applied in vitro (e.g., in vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours). At least 2-fold higher, preferably at least 3-fold, 4-fold, 5-fold, 6-fold or higher, in the population of DS-B cells.

[0118] In one embodiment, compared to HCT-116 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro, HCT-116 DS-B cells overexpress at least one or more of the following proteins: beta-2-glycoprotein 1, histone H2A type 1, poly(rC)-binding protein 2, kinectin, plectin, and NADH dehydrogenase [ubiquinone]1 alpha subcomplex subunit 7.

[0119] In one embodiment, HCT-116 DS-B cells differentially express at least one or more of the following proteins at detectable levels: beta-2-glycoprotein 1, histone H2A type 1, and poly(rC) binding protein 2, as compared to HCT-116 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied chemical stress.

[0120] In one embodiment, compared to HCT-116 cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro, HCT-116 DS-B cells overexpress at least one or more of the following membrane and / or cell surface proteins: mitochondrial phosphate carrier protein, integrin β4, lysosomal membrane glycoprotein 1, CD44 antigen, ribosome-binding protein 1, protein transport protein Sec61 subunit α isoform 1, kinectin, HLA class I histocompatibility complex A alpha chain, lysophospholipid acyltransferase 7, membrane-associated progesterone receptor component 1, microsomal glutathione S-transferase 1, NADH dehydrogenase [ubiquinone]1 β subcomplex subunit 4, desmoglein-2, integrin α3, tosin-1A-interacting protein 1, plasma membrane calcium transporter ATPase 1, mitochondrial phosphate carrier protein, integrin β4, lysosomal membrane glycoprotein 1, CD44 antigen, ribosome-binding protein 1, protein transporter Sec61 subunit α isoform 1, kinectin, HLA class I histocompatibility complex A alpha chain, lysophospholipid acyltransferase 7, membrane-associated progesterone receptor component 1, microsomal glutathione S-transferase 1, NADH dehydrogenase [ubiquinone]1 β subcomplex subunit 4, desmoglein-2, integrin α3, tosin-1A-interacting protein 1, plasma membrane calcium transporter ATPase 1, mitochondrial phosphate carrier protein, mitochondrial phosphate carrier protein, mitochondrial phosphate carrier protein, mitochondrial phosphate carrier protein, mitochondrial phosphate carrier protein, mitochondrial These proteins are involved in the regulation of mitochondrial function and function in the regulation of mitochondrial function. These proteins include mitochondrial ATPase 1, sphingosine-1-phosphate lyase 1, V-type proton ATPase 116 kDa subunit a1, mitochondrial inner membrane protein OXA1L, mitochondrial NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 5, transmembrane protein 43, amine oxidase [flavin-containing] B, protein transport protein Sec61 subunit beta, secretory carrier-associated membrane protein 3, protein FAM162A, retinol dehydrogenase 11, ADP-ribosylating factor-like protein 8B, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3, dolichol phosphate mannosyltransferase subunit 3, mitochondrial thiamine pyrophosphate carrier, and ORM1-like protein 2.

[0121] In one embodiment, the intermediate composition comprises or consists of (i) stressed LoVo cells, and (ii) stress and / or resistance proteins produced by these LoVo cells in response to: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a period ranging from about 6 hours to about 120 hours, preferably from about 24 hours to about 96 hours, e.g., 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours.

[0122] In one embodiment, the intermediate composition comprises or consists of (i) stressed LoVo cells, and (ii) stress and / or resistance proteins produced by these LoVo cells in response to: (i) in vitro culture in low serum culture conditions in 2% FBS medium, and (ii) In vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours. This intermediate composition is referred to herein as "LoVo DS-B."

[0123] In one embodiment, the LoVo DS-B comprises a stress and / or resistance protein selected from the group including or consisting of a radiation resistance protein, a heat stress resistance protein, a metabolic stress resistance protein, and combinations thereof.

[0124] In one embodiment, LoVo DS-B cells overexpress at least one or two of the following markers: CD243 (MDR-1) and CD66 (CEA); preferably, LoVo DS-B cells overexpress CD243 (MDR-1).

[0125] In one embodiment, LoVo DS-B cells express at least 10% more, preferably at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the markers CD243 (MDR-1) and CD66 (CEA), preferably the marker (MDR-1), compared to LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) chemical stress (e.g., in vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours); and / or compared to LoVo cells cultured in 2% FBS, and not subjected to in vitro applied chemical stress (e.g., in vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours). Additionally or alternatively, at least one or two of the following markers: CD243 (MDR-1) and CD66 (CEA), preferably CD243 (MDR-1), are expressed at least 10% more in a population of LoVo DS-B cells than a population of LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) chemical stress (e.g., in vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours), and / or than a population of LoVo cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied chemical stress (e.g., in vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours).Additionally or alternatively, the mean fluorescence intensity (MFI) of at least one or two of the following markers: CD243 (MDR-1) and CD66 (CEA), preferably CD243 (MDR-1), is at least 10% more, preferably at least 2-fold higher, preferably at least 3-fold, 4-fold, 5-fold, 6-fold or more higher, in a population of LoVo DS-B cells than a population of LoVo cells cultured in classical conditions, e.g., in 10% FBS, and not subjected to in vitro applied (i) metabolic stress, (ii) chemical stress (e.g., in vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours), and / or than a population of LoVo cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied chemical stress (e.g., in vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours).

[0126] In one embodiment, compared to LoVo cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro, LoVo DS-B cells overexpress at least one or more of the following proteins: beta-2-glycoprotein 1, histone H2A type 1, poly(rC) binding protein 2, kinectin, plectin, and NADH dehydrogenase [ubiquinone]1 alpha subcomplex subunit 7.

[0127] In one embodiment, LoVo DS-B cells differentially express at least one or more of the following proteins at detectable levels: beta-2-glycoprotein 1, histone H2A type 1, and poly(rC) binding protein 2, as compared to LoVo cells that have been subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to in vitro applied chemical stress.

[0128] In one embodiment, the LoVo cells are subjected to metabolic stress, e.g., cultured in 2% FBS, but not subjected to chemical stress applied in vitro, compared to LoVo cells. DS-B cells overexpress at least one or more of the following membrane and / or cell surface proteins: nuclear pore glycoprotein 210, reticulon-4, NADPH-cytochrome P450 reductase, long-chain fatty acid-CoA ligase 3, long-chain fatty acid-CoA ligase 4, vesicle-associated membrane protein-associated protein A, mitochondrial tricarboxylate transport protein, kinectin, vesicle-associated membrane protein-associated protein B / C, very long-chain (3R)-3-hydroxyacyl-CoA dehydratase 3, endoplasmic reticulum integral membrane protein VIP36, very long-chain enoyl-CoA reductase, microsomal glutathione S-transferase 1, cytochrome b5 type B, mitochondrial ATP synthase membrane subunit DAPIT, mitochondrial ATP synthase subunit f, cytochrome c oxidase subunit 2, mitochondrial heme protein cytochrome c oxidase subunit 2, mitochondrial heme protein cytochrome c oxidase subunit 3, mitochondrial heme protein cytochrome c oxidase subunit 1, mitochondrial heme protein cytochrome c oxidase subunit 2 ... Chrom c1, HLA class I histocompatibility complex B alpha chain, protein LYRIC, monocarboxylate transporter 1, cytochrome c oxidase subunit NDUFA4, mannosyl oligosaccharide glucosidase, signal peptidase complex catalytic subunit SEC11A, protein FAM162A, transmembrane emp24 domain-containing protein 2, retinol dehydrogenase 11, erlin-1, BRI3 binding protein, 3-beta-hydroxysteroid-delta(8),delta(7)-isomerase, receptor expression-enhancing protein 5, CDGSH iron-sulfur domain-containing protein 2, CD9 antigen, extended synaptotagmin-2, very long-chain acyl-CoA synthetase, Kunitz-type protease inhibitor 2, CD166 antigen, long-chain fatty acid-CoA ligase 1, leukocyte surface antigen surface antigen CD47, tapasin, and beta-1,3-galactosyl-O-glycosyl glycoprotein β-1,6-N-acetylglucosaminyltransferase 3.

[0129] Manufacturing method The present invention also relates to a method for producing the intermediate composition. In one embodiment, the method for preparing the intermediate composition described above comprises the steps of: a) culturing HT-29, HCT-116 or LoVo cells in a suitable medium; b) subjecting the HT-29, HCT-116 or LoVo cells cultured in step a) to one or more stresses in vitro, and in response to the one or more stresses, these HT-29, HCT-116 or LoVo cells develop resistance mechanisms, thereby producing stress and / or resistance proteins; and c) recovering the stressed HT-29, HCT-116 or LoVo cells together with the stress and / or resistance proteins that they produced in step b). Includes.

[0130] In one embodiment, step a) is carried out in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5). In one embodiment, step a) is carried out under low serum culture conditions in 2% FBS medium. In one embodiment, this culture in the depleted medium is maintained during step b). The stress applied in step b) has been described previously.

[0131] In one embodiment, step c) is performed at least several hours after the completion of step b), preferably 12, 24, 36, 48, 60, 72, 84, 96 hours or more after the completion of step b), ensuring that the HT-29, HCT-116 or LoVo cells have sufficient time to develop their resistance mechanisms in response to one or more stresses, and thus to produce stress and / or resistance proteins.

[0132] In one embodiment, the method further comprises a step d) of treating the stressed HT-29, HCT-116 or LoVo cells and the stress and / or resistance proteins they produce, all together collected in step c), with a molecule or means capable of rendering said stress and / or resistance proteins immunogenic.

[0133] In one embodiment, step d) comprises or consists of linking or complexing the stress and / or resistance protein to a means capable of conferring immunogenicity.

[0134] The means capable of imparting immunogenicity are well known to those skilled in the art.In one embodiment, the means capable of imparting immunogenicity comprises or consists of one or more molecules that do not naturally exist in HT-29, HCT-116 and LoVo cells or in their environment.Such means are described in detail above, and include but are not limited to haptens.

[0135] Examples of haptens include, but are not limited to, 2,4-dinitrophenyl (DNP); 2,4-dinitrofluorobenzene; sulfanilic acid; N-iodoacetyl-N'-(5-sulfonic acid-naphthyl)ethylenediamine; aniline; p-aminobenzoic acid; biotin; fluorescein and its derivatives (FITC, TAMRA, and Texas Instruments). Red); digoxigenin; 5-nitro-3-pyrazolecarbamide; 4,5-dimethoxy-2-nitrocinamide; 2-(3,4-dimethoxyphenyl)-quinoline-4-carbamide; 2,1,3-benzoxadiazole-5-carbamide; 3-hydroxy-2-quinoxalinecarbamide; 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL); rotenone isoxazoline; (E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenodi)acetamide; 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid; 2-acetamido-4-methyl-5-thiazolesulfonamide; and p-methoxyphenylpyrazopodophilamide.

[0136] In one embodiment, step d) comprises or consists of haptenizing the stress and / or resistance protein. In one embodiment, step d) comprises or consists of 2,4-dinitrophenylating the stress and / or resistance protein (i.e. haptenizing the stress and / or resistance protein with 2,4-dinitrophenyl). Those skilled in the art are familiar with means and methods for haptenizing proteins, such as stress and / or resistance proteins.

[0137] In one embodiment, where the intermediate composition further comprises tumor-associated antigens (TAA) and / or tumor-specific antigens (TSA) specific for HT-29, HCT-116 or LoVo cells, step d) also comprises linking or complexing these TAAs and / or TSAs to a means capable of conferring immunogenicity, under the same conditions as described above for stress and / or resistance proteins.

[0138] In one embodiment, the method further comprises a step of inactivating HT-29, HCT-116 or LoVo cells to render them non-proliferative. If applicable, this step can be performed any time after HT-29, HCT-116 or LoVo cells have developed their resistance mechanisms in response to stresses applied in vitro, and thus after HT-29, HCT-116 or LoVo cells have produced stress and / or resistance proteins. In one embodiment, the step of inactivating HT-29, HCT-116 or LoVo cells is performed at least several hours after the completion of step b), preferably 12, 24, 36, 48, 60, 72, 84, 96 hours or more after the completion of step b). In one embodiment, the step of inactivating HT-29, HCT-116 or LoVo cells is performed after the completion of step c). In one embodiment, the step of inactivating the HT-29, HCT-116 or LoVo cells, if applicable, is carried out after completion of step d).

[0139] Those skilled in the art are familiar with the means and methods of rendering cells non-proliferative. For example, cells can be rendered non-proliferative by irradiation at a dose sufficient to kill or inactivate the cells. In one embodiment, irradiation comprises or consists of irradiating the cells with a total dose of 25 Gy or more to render the cells non-proliferative. Alternatively or additionally, cells can be rendered non-proliferative by ethanol fixation, for example using about 10% to about 50% v / v ethanol. Alternatively or additionally, cells can be rendered non-proliferative, for example by at least one freeze-thaw cycle. Alternatively or additionally, cells can be rendered non-proliferative by linking or complexing to a means capable of rendering the cells immunogenic, for example by haptenization.

[0140] Those skilled in the art are also familiar with the means and methods of determining whether cells are non-proliferative or not, for example, by performing viability tests, by cell culture (to assess complete lack of proliferation) and / or by propidium iodide (to distinguish between live and dead cells).

[0141] In one embodiment, the method is for the production of an intermediate composition "HT-29 DS-A" comprising or consisting of (i) stressed HT-29 cells and (ii) stress and / or resistance proteins produced by these HT-29 cells in response to in vitro applied (i) metabolic stress, (ii) radiation and (iii) heat stress.

[0142] In this embodiment, step b) of the method comprises or consists of subjecting the HT-29 cells cultured in step a) of the method in vitro to the following stresses: metabolic stress, irradiation and heat stress. Irradiation, heat stress and metabolic stress have been described previously.

[0143] In this embodiment, step b) of the method comprises or consists of subjecting the HT-29 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) irradiation with a total dose ranging from about 0.25 to about 25 Gy, preferably from about 1 to about 15 Gy, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gy (in one embodiment, the irradiation time is in the range of about 1 to about 20 minutes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably in the range of about 1 to about 5 minutes); and (iii) In vitro heat shock at a temperature in the range of about 38° C. to about 45° C., e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., applied to the cells for a period of about 15 to about 4 hours, preferably about 30 minutes to about 2 hours, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

[0144] In this embodiment, step b) of the method comprises or consists of subjecting the HT-29 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation for about 1 to about 5 minutes with a total dose of 10 Gy; and (iii) In vitro heat shock at a temperature of about 42° C. for about 60 minutes.

[0145] In one embodiment, the stress according to (i) is applied for the entire duration of step b), in one embodiment, the stress according to (iii) is applied immediately or shortly after the stress according to (ii), for example, less than 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, or 3 hours after the end of the stress according to (ii) while maintaining the stress according to (i).

[0146] In one embodiment, the method is for the production of an intermediate composition "HCT-116 DS-A" comprising or consisting of (i) stressed HCT-116 cells and (ii) stress and / or resistance proteins produced by these HCT-116 cells in response to in vitro applied (i) metabolic stress, (ii) irradiation and (iii) heat stress. In this embodiment, step b) of the method comprises or consists of subjecting in vitro the HCT-116 cells cultured in step a) of the method to the following stresses: metabolic stress, irradiation and heat stress. Irradiation, heat stress and metabolic stress have been described previously.

[0147] In this embodiment, step b) of the method comprises or consists of subjecting the HCT-116 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) irradiation with a total dose ranging from about 0.25 to about 25 Gy, preferably from about 1 to about 15 Gy, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gy (in one embodiment, the irradiation time is in the range of about 1 to about 20 minutes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably in the range of about 1 to about 5 minutes); and (iii) In vitro heat shock at a temperature in the range of about 38° C. to about 45° C., e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., applied to the cells for a period of about 15 to about 4 hours, preferably about 30 minutes to about 2 hours, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes. In this embodiment, step b) of the method comprises or consists of subjecting the HCT-116 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation for about 1 to about 5 minutes with a total dose of 10 Gy; and (iii) In vitro heat shock at a temperature of about 42° C. for about 60 minutes.

[0148] In one embodiment, the stress according to (i) is applied for the entire duration of step b), in one embodiment, the stress according to (iii) is applied immediately or shortly after the stress according to (ii), such as 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, or 3 hours after the end of the stress according to (ii) while maintaining the stress according to (i).

[0149] In one embodiment, the method is for the production of an intermediate composition "LoVo DS-A" comprising or consisting of (i) stressed LoVo cells and (ii) stress and / or resistance proteins produced by these LoVo cells in response to in vitro applied (i) metabolic stress, (ii) radiation and (iii) heat stress.

[0150] In this embodiment, step b) of the method comprises or consists of subjecting the LoVo cells cultured in step a) of the method to the following stresses in vitro: metabolic stress, irradiation and heat stress. Irradiation, heat stress and metabolic stress have been described previously.

[0151] In this embodiment, step b) of the method comprises or consists of subjecting the LoVo cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) irradiation with a total dose ranging from about 0.25 to about 25 Gy, preferably from about 1 to about 15 Gy, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gy (in one embodiment, the irradiation time is in the range of about 1 to about 20 minutes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, preferably in the range of about 1 to about 5 minutes); and (iii) In vitro heat shock at a temperature in the range of about 38° C. to about 45° C., e.g., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C., applied to the cells for a period of about 15 to about 4 hours, preferably about 30 minutes to about 2 hours, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

[0152] In this embodiment, step b) of the method comprises or consists of subjecting the LoVo cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation for about 1 to about 5 minutes with a total dose of 10 Gy; and (iii) In vitro heat shock at a temperature of about 42° C. for about 60 minutes.

[0153] In one embodiment, the stress according to (i) is applied for the entire duration of step b), in one embodiment, the stress according to (iii) is applied immediately or shortly after the stress according to (ii), such as 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, or 3 hours after the end of the stress according to (ii) while maintaining the stress according to (i).

[0154] In one embodiment, the method is for the production of an intermediate composition "HT-29 DS-B" comprising or consisting of (i) stressed HT-29 cells and (ii) stress and / or resistance proteins produced by these HT-29 cells in response to (i) metabolic stress and (ii) chemical stress applied in vitro.

[0155] In this embodiment, step b) of the method comprises or consists of subjecting the HT-29 cells cultured in step a) of the method to the following stresses in vitro: metabolic stress, and chemical stress. Chemical and metabolic stresses have been described previously.

[0156] In this embodiment, step b) of the method comprises or consists of subjecting the HT-29 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a period ranging from about 6 hours to about 120 hours, preferably from about 24 hours to about 96 hours, e.g., 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours.

[0157] In this embodiment, step b) of the method comprises or consists of subjecting the HT-29 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) In vitro exposure to about 13 μM oxaliplatin for about 72 hours.

[0158] In one embodiment, the stress according to (i) is applied for the entire duration of step b), In one embodiment, the stress according to (ii) is applied while maintaining the stress according to (i).

[0159] In one embodiment, the method is for the production of an intermediate composition "HCT-116 DS-B" comprising or consisting of (i) stressed HCT-116 cells and (ii) stress and / or resistance proteins produced by these HCT-116 cells in response to (i) metabolic stress and (ii) chemical stress applied in vitro.

[0160] In this embodiment, step b) of the method comprises or consists of subjecting the HCT-116 cells cultured in step a) of the method to the following stresses in vitro: metabolic stress, and chemical stress. Chemical and metabolic stresses have been described previously.

[0161] In this embodiment, step b) of the method comprises or consists of subjecting the HCT-116 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a period ranging from about 6 hours to about 120 hours, preferably from about 24 hours to about 96 hours, e.g., 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours.

[0162] In this embodiment, step b) of the method comprises or consists of subjecting the HCT-116 cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) In vitro exposure to about 31.5 nM, 100 nM or 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours. In one embodiment, the stress according to (i) is applied for the entire duration of step b), In one embodiment, the stress according to (ii) is applied while maintaining the stress according to (i).

[0163] In one embodiment, the method is for the production of an intermediate composition "LoVo DS-B" comprising or consisting of (i) stressed LoVo cells and (ii) stress and / or resistance proteins produced by these LoVo cells in response to (i) metabolic stress and (ii) chemical stress applied in vitro.

[0164] In this embodiment, step b) of the method comprises or consists of subjecting the LoVo cells cultured in step a) of the method to the following stresses in vitro: metabolic stress, and chemical stress. Chemical stress and metabolic stress have been described above.

[0165] In this embodiment, step b) of the method comprises or consists of subjecting the LoVo cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in a depleted medium (e.g., a medium partially or completely lacking one or more substances useful or even essential for the growth and / or survival of cancer cells), under low oxygen and / or at low pH (e.g., below pH 6.5); (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a period ranging from about 6 hours to about 120 hours, preferably from about 24 hours to about 96 hours, e.g., 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, or 96 hours.

[0166] In this embodiment, step b) of the method comprises or consists of subjecting the LoVo cells cultured in step a) of the method to the following in vitro stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) In vitro exposure to about 5 μM fluorouracil (5-FU) for about 48 hours. In one embodiment, the stress according to (i) is applied for the entire duration of step b), In one embodiment, the stress according to (ii) is applied while maintaining the stress according to (i).

[0167] The present invention also relates to a method for producing a composition comprising or consisting of the above-mentioned (i) stressed HT-29, HCT-116 and LoVo cells, and (ii) immunogenic stress and / or resistance proteins, the final composition being referred to herein as "DP."

[0168] In one embodiment, a method for producing a composition comprising or consisting of (i) stressed HT-29, HCT-116 and LoVo cells and (ii) immunogenic stress and / or resistance proteins as described above comprises the following steps: a) obtaining HT-29 DS-A, HCT-116 DS-A, LoVo DS-A, HT-29 DS-B, HCT-116 DS-B and LoVo DS-B intermediate compositions using the above-mentioned method for producing the intermediate compositions; and b) mixing together the HT-29 DS-A, HCT-116 DS-A, LoVo DS-A, HT-29 DS-B, HCT-116 DS-B and LoVo DS-B intermediate compositions. Includes.

[0169] In one embodiment, step b) comprises dissolving six intermediate compositions in about 10 mL of composition. 6 ~about 10 9 10 stressed HT-29, HCT-116 and LoVo cells, preferably about 10 per mL of the composition.6 ~about 10 8 Stressed HT-29, HCT-116 and LoVo cells, for example, about 1x10 per mL of composition 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , or 1x10 8 In one embodiment, step b) comprises mixing the six intermediate compositions with a final concentration of about 3x10 cells / mL of the composition. 7 Mix with a final concentration of 100 mM stressed HT-29, HCT-116 and LoVo cells.

[0170] In one embodiment, step b) comprises mixing the six intermediate compositions in equal ratios with stressed HT-29, HCT-116 and LoVo cells (i.e., about 1:1:1:1:1:1:1). In one embodiment, step b) comprises mixing the six intermediate compositions in a ratio comprising or consisting of at least 1.5, 2 or 2.5 times more stressed HT-29 than stressed HCT-116 or LoVo cells. In one embodiment, step b) comprises mixing the six intermediate compositions in a ratio comprising or consisting of at least 1.5, 2 or 2.5 times more stressed HCT-116 than stressed HT-29 or LoVo cells. In one embodiment, step b) comprises mixing the six intermediate compositions in a ratio comprising or consisting of at least 1.5, 2 or 2.5 times more stressed LoVo cells than stressed HT-29 or HCT-116 cells.

[0171] In one embodiment, step b) comprises mixing together: -about 10 5 ~about 10 810 stressed HT-29 cells (derived from HT-29 DS-A and HT-29 DS-B), preferably about 10 6 ~about 10 7 10 stressed HT-29 cells, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x10 7 HT-29 cells subjected to stress, -about 10 5 ~about 10 8 10 stressed HCT-116 cells (derived from HCT-116 DS-A and HCT-116 DS-B), preferably about 10 6 ~about 10 7 10 stressed HCT-116 cells, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x10 7 stressed HCT-116 cells, and -about 10 5 ~about 10 8 10 stressed LoVo cells (LoVo DS-A and LoVo DS-B), preferably about 10 6 ~about 10 7 Stressed LoVo cells, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x107 LoVo cells subjected to individual stresses.

[0172] Therapeutic Uses and Methods The present invention also relates to a method of treating cancer in a subject in need thereof, comprising administering to said subject a composition comprising or consisting of (i) stressed HT-29, HCT-116 and LoVo cells and (ii) immunogenic stress and / or resistance proteins produced by these cells in response to stress applied in vitro, as defined herein above.

[0173] The present invention also relates to a composition comprising or consisting of (i) stressed HT-29, HCT-116 and LoVo cells and (ii) immunogenic stress and / or resistance proteins produced by these cells in response to stress applied in vitro, as defined herein above, for use in the treatment of cancer in a subject in need thereof.

[0174] The present invention also relates to the use of a composition comprising or consisting of (i) stressed HT-29, HCT-116 and LoVo cells and (ii) immunogenic stress and / or resistance proteins produced by these cells in response to stress applied in vitro, as defined herein above, for the treatment of cancer in a subject in need thereof.

[0175] The present invention also relates to the use of a composition comprising or consisting of (i) stressed HT-29, HCT-116 and LoVo cells and (ii) immunogenic stress and / or resistance proteins produced by these cells in response to stress applied in vitro, as defined herein above, for the manufacture of a medicament for the treatment of cancer in a subject in need thereof. In one embodiment, treating cancer comprises eliciting an immune response against cancer cells from said cancer.

[0176] Examples of cancers include those listed in the International Statistical Classification of Diseases and Related Health Problems (ICD), 10th Revision, Chapter II, Blocks C00-D48. Further examples of cancer include, but are not limited to, recurrent, metastatic or multidrug resistant cancers. Further examples of cancer include, but are not limited to, adenofibroma, adenoma, primary myelofibrosis, AIDS-related malignancies, ameloblastoma, anal cancer, angiocystic mediastinal lymph node hyperplasia, angiokeratoma, eosinophilic angiolymphocytosis, hemangiomatosis, anhidrotic ectodermal dysplasia, anterior and posterior dysplasia, apocrine metaplasia, apdoma, asphyxiating thoracic dysplasia, astrocytoma (including, for example, cerebellar astrocytoma and cerebral astrocytoma), atrial digital dysplasia, atypical melanocytic hyperplasia, atypical dysplasia, autologous parenchymal metaplasia, basal cell hyperplasia, benign Giant lymphadenopathy, bile duct cancer (including, for example, extrahepatic bile duct cancer), bladder cancer, bone cancer, brain tumors (including, for example, brain stem glioma, cerebellar astrocytoma glioma, malignant glioma, supratentorial primitive neuroectodermal tumor, optic pathway and hypothalamic glioma, ependymoma, medulloblastoma, gestational trophoblastic tumor glioma, and paraganglioma), branchiomas, female breast cancer, male breast cancer, bronchial adenoma / carcinoid, bronchopulmonary dysplasia, cancerous growths of epithelial cells, precancerous growths of epithelial cells, metastatic growths of epithelial cells, carcinoid heart disease, Tumors (including, for example, gastrointestinal carcinoid tumors), carcinomas (including, for example, carcinoma of unknown primary site, adrenal cortical carcinoma, pancreatic islet cell carcinoma, adenocarcinoma, adrenal cortical carcinoma, basal cell carcinoma, basal squamous cell carcinoma, bronchiolar carcinoma, Brown-Pearce carcinoma, cystadenocarcinoma, ductal carcinoma, hepatocellular carcinoma, Krebs carcinoma, papillary carcinoma, oat cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, squamous cell carcinoma, transitional cell carcinoma, Walker carcinoma, Merkel cell carcinoma, and skin carcinoma), cementoma, cementum hyperplasia, cerebral dysplasia, cervical cancer, cervical dysplasia, cholangiocarcinoma ... blastoma, chondroectodermal dysplasia, chordoma, chiroma, chrondroma, cleidocranial dysplasia, colon adenocarcinoma, colon cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, colorectal carcinoma, regional metastatic colorectal cancer, congenital adrenal hyperplasia, congenital ectodermal dysplasia, congenital sebaceous hyperplasia, connective tissue dysplasia, craniocarpal tarsal dysplasia, cranioshaft dysplasia, craniometaphyseal dysplasia, craniopharyngioma, cystoma, cystadenoma, cystic hyperplasia (including, for example, cystic hyperplasia of the breast), supralobular cystosarcoma phyllodes), dentin dysplasia, denture fibrosis, diaphyseal dysplasia, ductal hyperplasia, dysgerminoma, unilateral epiphyseal dysplasia, multiple epiphyseal dysplasia, epiphyseal diastema dysplasia, dysplasia epiphysialis punctata, ectodermal dysplasia,Ehrlich's tumor, ameloblastoma, cerebro-ocular hypoplasia, endometrial cancer (including, for example, ependymoma and endometrial hyperplasia), ependymoma, epithelial carcinoma, epithelial dysplasia, epithelial metaplasia, esophageal cancer, Ewing's family of tumors (including, for example, Ewing's sarcoma), extrahepatic bile duct cancer, eye cancer (including, for example, intraocular melanoma and retinoblastoma), facial-digital dysplasia, familial fibrous jaw dysplasia, familial white fold dysplasia, fibroma, fibromuscular dysplasia, fibromuscular hyperplasia, fibrous dysplasia, florescent dysplasia, focal epithelial thickening, bladder cancer, ganglioneuroma, stomach cancer (including, for example, stomach cancer), gastrointestinal carcinoid tumor, gastrointestinal cancer, gastrointestinal tumor, Gaucher disease, germ cell tumors (including, for example, extracranial germ cell tumors, extragonadal germ cell tumors, and ovarian germ cell tumors), giant cell tumor, gingival hyperplasia, glioblastoma, glomus hemangioma, granulosa cell tumor, hemispheric ovarian tumor, hamartoma, head and neck cancer, hemangioendothelioma, hemangioma, hemangiopericytoma, hepatocellular carcinoma, hepatoma, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia Common diseases, histiocytoma, histiocytosis, hypergammaglobulinemia, hypohidrotic ectodermal dysplasia, hypopharyngeal cancer, inflammatory fibrous gingival hyperplasia, inflammatory papillary gingival hyperplasia, intestinal cancer, intestinal metaplasia, intestinal polyps, intraocular melanoma, intravascular papillary endothelial hyperplasia, kidney cancer, throat cancer, leiomyosarcoma, leukemia (e.g. acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute hairy cell leukemia, acute myeloid leukemia, acute myelogenous leukemia, acute myelogenous leukemia, acute hairy cell leukemia, acute myelogenous ... leukemia, acute B-cell leukemia, acute T-cell leukemia, HTLV leukemia, chronic lymphocytic leukemia, chronic lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelogenous leukemia, chronic hairy cell leukemia, chronic B-cell leukemia, chronic T-cell leukemia, and chronic HTLV leukemia), Leydig cell tumor, lip and oral cavity cancer, lipoma, liver cancer, lung cancer (including, for example, small cell lung cancer and non-small cell lung cancer), ductal leukemia , lymphangioma, lymphoma (e.g., including AIDS-related lymphoma, central nervous system lymphoma, primary central nervous system lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma during pregnancy, mast cell lymphoma, B cell lymphoma, adenolymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, large cell lymphoma, and small cell lymphoma), lymphocytopenic thymic dysplasia, lymphoproliferative disorders, macroglobulinemia (e.g.,Waldenstrom's macroglobulinemia), malignant carcinoid syndrome, malignant mesothelioma, malignant thymoma, breast dysplasia, mandibulofacial dysplasia, medulloblastoma, meningioma, mesenchymoma, mesothelioma (including malignant mesothelioma), metaphyseal dysplasia, metaplastic anemia, deformed ossification, metaplastic polyp, Metastatic cervical squamous cell carcinoma (including, for example, metastatic cervical squamous cell carcinoma of unknown primary), Mondini dysplasia, monoostotic fibrous dysplasia, mucosal epithelial dysplasia, multiple endocrine neoplasia syndrome, multiple epiphyseal dysplasia, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloid metaplasia, myeloproliferative disorders, chronic myeloproliferative disorders, myoblastoma, fibroids, myxoma, nasal and paranasal cancer, nasopharyngeal cancer, prostate tumor, colon tumor, abdominal tumor, bone tumor, breast tumor, gastrointestinal tumor, liver tumor, pancreatic tumor, peritoneal tumor, endocrine tumor ( For example, tumors of the adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, and thyroid gland, eye tumors, head and neck tumors, tumors of the nervous system (including, for example, central nervous system and peripheral nervous system tumors), lymphatic system tumors, pelvic tumors, skin tumors, soft tissue tumors, splenic tumors, breast tumors, tumors of the genitourinary tract, schwannoma, neuroblastoma, neuroepithelioma, neurofibroma, neurofibromatosis, neuroma, nodular prostatic hyperplasia, nodular regenerative hyperplasia, oculo-auricular dysplasia, oculo-dentofacial dysplasia, oculo-spinal dysplasia, dental dysplasia, odontoma, Oculomandibular dysplasia, oropharyngeal cancer, osteoma, ovarian cancer (e.g., including ovarian epithelial carcinoma and ovarian tumor of low malignant potential), pancreatic cancer (e.g., including islet cell pancreatic cancer and exocrine pancreatic cancer), papilloma, paraganglioma, nonchromaffin paraganglioma, paranasal sinus and nasal cancer, dysproteinemia, parathyroid carcinoma, apical cemental dysplasia, pheochromocytoma (e.g., including penile cancer), pineal and supratentorial primitive neuroectodermal tumors, pinealoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, plasmacytoma, pleuropulmonary blastoma, polyostotic adenocarcinoma fibrous dysplasia, polyps, cancer of pregnancy, preneoplastic disorders (e.g., benign proliferative disorders including, e.g., benign tumors, fibrocystic conditions, tissue hyperplasia, intestinal polyps, colonic polyps, esophageal dysplasia, leukoplakia, keratosis, Bowen's disease, farmer's skin, actinic cheilitis, and actinic keratosis), primary hepatocellular carcinoma, primary liver cancer, primary myeloid metaplasia, prostate cancer, pseudoachondroplasia spondyloepiphyseal dysplasia, pseudoepitheliomatous proliferation, purpura, rectal cancer, renal cancer (e.g., kidney cancer, renal pelvis, ureter cancer, transitional cell carcinoma of the renal pelvis and ureter,reticuloendotheliosis, retinal dysplasia, retinoblastoma, salivary gland cancer, sarcoma (e.g., uterine sarcoma, soft tissue sarcoma, carcinosarcoma, chondrosarcoma, fibrosarcoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, rhabdomyosarcoma, sarcoidosis sarcoma, osteosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone, and clear cell sarcoma of tendon sheath), sclerosing hemangioma, secondary myeloid metaplasia, senile sebaceous hyperplasia, septo-optic dysplasia, Sertoli cell tumor, Sezary syndrome, skin cancer (e.g., melanoma skin cancer and non-melanoma skin cancer), cancer), small intestine cancer, spondyloepiphyseal dysplasia, squamous metaplasia (e.g., including squamous metaplasia of the amniotic membrane), gastric cancer, supratentorial primitive neuroectodermal and pineal tumors, supratentorial primitive neuroectodermal tumors, symptomatic myeloid metaplasia, teratoma, testicular cancer, thecocytoma, thymoma (e.g., including malignant thymoma), thyroid cancer, trophoblastic tumors (e.g., including gestational trophoblastic tumors), ureteral cancer, urethral cancer, uterine cancer, vaginal cancer, ventricular radial dysplasia, verrucous membrane hyperplasia, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor. In one embodiment, the cancer is selected from the group comprising or consisting of renal cancer, liver cancer, pancreatic cancer, endometrial cancer, colorectal cancer, ovarian cancer, breast cancer, lung cancer, head and neck cancer, cervical cancer, melanoma and glioma. In one embodiment, the cancer is selected from the group including or consisting of colon cancer and colorectal cancer, including but not limited to colon adenocarcinoma, colon cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, and colorectal cancer.

[0177] In one embodiment, the composition is or will be administered once, hi one embodiment, the composition is or will be administered several times, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times. In one embodiment, the composition is administered or will be administered weekly, every two weeks, every three weeks, monthly, every two months, every three months, every six months, or yearly. In one embodiment, as an induction or bolus regimen, it is or will be administered, preferably over the first few weeks, e.g., the first 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more, preferably over the first 6-8 weeks, weekly, every two weeks, every three weeks, monthly, preferably weekly. In one embodiment, the composition is or will be administered monthly, every two months, every three months, every six months, or yearly as a booster regimen following an induction or bolus regimen. In one embodiment, the composition is administered or will be administered weekly for the first 6-8 weeks; then about 1 month later; then about 3 months later; and then every 6 months until the cancer is treated. In one embodiment, the composition comprises about 10 5 ~about 10 8 HT-29, HCT-116 and LoVo cells subjected to stress ranging from about 10 to about 50 μg / ml. 6 ~about 10 7 Stressed HT-29, HCT-116 and LoVo cells in the range of 1x10, e.g., approximately 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , or 1x10 7 A total dose of 1000 mg of stressed HT-29, HCT-116 and LoVo cells has been or will be administered. In one embodiment, the composition is or will be administered as a single dose. In one embodiment, the composition is or will be administered in at least two doses, for example, 2, 3, 4, 5 or more doses. In one embodiment, the composition comprises about 3×10 6In one embodiment, the composition is administered or will be administered at a total dose of about 6x10 stressed HT-29, HCT-116 and LoVo cells. 6 In one embodiment, the composition is administered or will be administered at a total dose of 6x10 stressed HT-29, HCT-116 and LoVo cells. 6 A single dose of stressed HT-29, HCT-116 and LoVo cells, or approximately 3x10 6 It has been or will be administered in two doses of each of stressed HT-29, HCT-116 and LoVo cells. In one embodiment, the composition is administered or will be administered in a volume ranging from about 10 μL to about 1 mL, e.g., about 10 μL, about 50 μL, about 100 μL, about 200 μL, about 300 μL, about 400 μL, about 500 μL, about 600 μL, about 700 μL, about 800 μL, about 900 μL, or 1 mL, preferably about 100 μL. In one embodiment, the composition is or will be administered systemically, hi one embodiment, the composition is or will be administered by injection, preferably by systemic injection.

[0178] Examples of systemic injection include, but are not limited to, intravenous (iv), subcutaneous (sq), intradermal (id), intramuscular (im), intra-arterial, parenteral, intranodal, intraperitoneal (ip), intracardiac, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intranasal, intratumoral (it), intravesicular, and perfusion. In one embodiment, the composition is or will be administered intradermally, subcutaneously, intratumorally or intravenously. In one embodiment, the composition is or will be administered intradermally. Other suitable routes of administration are likewise contemplated by the present invention, and the mode of administration will ultimately be decided by the attending physician within the scope of sound medical judgment.

[0179] In one embodiment, the composition is or will be administered to the subject prior to, concurrently with, or following administration of at least one additional therapy.

[0180] Examples of suitable additional therapies include chemotherapeutic agents, radiation therapy and immune stimulants as defined above. Suitable examples of radiation therapy include, but are not limited to, external beam radiation therapy (e.g., superficial x-ray therapy, orthovoltage x-ray therapy, megavoltage x-ray therapy, radiosurgery, stereotactic radiotherapy, cobalt therapy, electron therapy, fast neutron therapy, neutron capture therapy, proton beam therapy, etc.); brachytherapy; unsealed source radiation therapy; tomotherapy, etc.

[0181] Suitable examples of chemotherapeutic agents include those described in subgroup L03 of the Anatomical Therapeutic Classification of Cancer. Further examples of immune stimulants include, but are not limited to, cytokines (e.g., filgrastim, pegfilgrastim, lenograstim, molgramostim, sargramostim, ancestim, albinterferon, interferon alpha natural, interferon alpha interferon alpha 2a, peginterferon alpha 2a, interferon alpha 2b, interferon alpha n1, interferon alphacon-1, interferon alpha-n3, interferon beta natural, interferon beta 1b, interferon gamma, aldesleukin, oprelvekin, etc. ); immune checkpoint inhibitors (e.g., inhibitors of CTLA4, PD-1, PD-L1, LAG-3, B7-H3, B7-H4, TIM3, A2AR, and / or IDO, including nivolumab, pembrolizumab, pidilizumab, AMP-224, MPDL3280A, MDX-1105, MEDI-4736, allelumab, ipilimumab, tremelimumab, pidilizumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, mogamulizumab, varlilumab, avelumab, galiximab, AMP-514, AUNP 12, indoximod, NLG-919, INCB024360, etc.);Toll-like receptor agonists (e.g., buprenorphine, carbamazepine, ethanol, fentanyl, GS-9620, imiquimod, lefitolimod, levorphanol, methadone, morphine, (+)-morphine, morphine-3-glucuronide, oxcarbazepine, oxycodone, pethidine, resiquimod, SD-101, tapentadol, tilsotolimod, VTX-2337, Cryptococcus-derived glucuronoxylomannan, Mycoplasma-derived MALP-2, Mycoplasma-derived MALP-404, Borrelia-derived OspA, Neisseria or Haemophilus-derived porin, hsp60, hemagglutinin , LcrV from Yersinia, bacterial flagellin, lipopolysaccharide, lipoteichoic acid, lipomannan from Mycobacterium, glycosylphosphatidylinositol, lysophosphatidylserine, lipophosphoglycan from Leishmania, zymosan from Saccharomyces, Pam2CGDPKHPKSF, Pam3CSK4, CpG oligodeoxynucleotides, poly(I:C) nucleic acid sequences, poly(A:U) nucleic acid sequences, STING receptor agonists (e.g., those described in §0240 of WO2017100305, vadimesan, CL656, ADU-S100, 3'3'-cGAMP, 2'3'-cGAMP, ML RR-S2 CDG, ML RR-S2 cGAMP, cyclic di-GMP, DMXAA, DiABZI, etc.);CD1 ligand;growth hormone;granulocyte-macrophage colony-stimulating factor (GM-CSF);immunocyanin;pegademase;prolactin;tasonermin;female sex steroid hormones;histamine dihydrochloride;polyICLC;vitamin D;lentinan;plerixafor;roquinimex;mifamurtide, glatiramer acetate;thymopentin;thymosin alpha 1;thymulin;polyinosinic (acid) polycytidylic acid;pidotimod;bacillus Calmette-Guerin;melanoma vaccine;sipuleucel-T;etc.;

[0182] In one embodiment, the composition may be administered to the subject following administration of a chemotherapeutic agent, such as, for example, cyclophosphamide. Additionally or alternatively, the composition may be administered to the subject simultaneously with administration of an immune stimulant, such as, for example, granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0183] Kit of parts The present invention also relates to a kit of parts. The term "kit of parts", also referred to as a "package", "commercial package" or "pharmaceutical package", encompasses physically separated component entities that are intended to be stored and / or used separately but functionally related to one another. In particular, the term "kit of parts" encompasses physically separated component substances that can be used or administered separately in any order, typically but not necessarily within a given time interval ranging from hours to days, weeks or longer; or simultaneously as an extemporaneous preparation, or individually in any order, within a time interval ranging from seconds to minutes or hours.

[0184] In one embodiment the kit-of-parts comprises: - an intermediate composition as defined above designated "HT-29 DS-A"; - an intermediate composition as defined above, designated "HCT-116 DS-A"; and - an intermediate composition as defined above, designated "LoVo DS-A"

[0185] In one embodiment the kit-of-parts comprises: - an intermediate composition as defined above designated "HT-29 DS-B"; - an intermediate composition as defined above, designated "HCT-116 DS-B"; and - an intermediate composition as defined above designated "LoVo DS-B"

[0186] In one embodiment the kit-of-parts comprises: - an intermediate composition as defined above designated "HT-29 DS-A"; - an intermediate composition as defined above, designated "HCT-116 DS-A"; - an intermediate composition as defined above, designated "LoVo DS-A"; - an intermediate composition as defined above designated "HT-29 DS-B"; - an intermediate composition as defined above, designated "HCT-116 DS-B"; and - an intermediate composition as defined above designated "LoVo DS-B"

[0187] In one embodiment, the kit-of-parts further comprises instructions for mixing intermediate compositions according to the method for producing the above-mentioned composition (designated "DP") comprising or consisting of (i) stressed HT-29, HCT-116 and LoVo cells, and (ii) immunogenic stress and / or resistance proteins described above. In one embodiment the kit-of-parts further comprises at least one additional therapeutic agent, such as, for example, at least one chemotherapeutic agent and / or immunostimulant, as defined above.

[0188] In one embodiment the kit-of-parts comprises: - the composition as defined above designated "DP"; - at least one additional therapeutic agent, such as, for example, at least one chemotherapeutic agent and / or immune stimulant, as defined above. [Brief description of the drawings]

[0189] [Figure 1A] Two Venn diagrams comparing the number of proteins identified in each of the three human cell lines (HT-29, HCT-116 and LoVo) relative to untreated samples. Figure 1A: RCB sample. Figure 1B: MCB sample. [Figure 1B] Two Venn diagrams comparing the number of proteins identified in each of the three human cell lines (HT-29, HCT-116 and LoVo) relative to untreated samples. Figure 1A: RCB sample. Figure 1B: MCB sample. [Diagram 2]Graph showing the difference in the number of expressed proteins in HCT-116 MCB, DS-A, DS-B, and the final product (DP) containing all six DSs (hence containing HCT-116 DS-A and DS-B) compared to HCT-116 RCB cultured in classical conditions (containing 10% FBS). "+" indicates newly expressed proteins and "-" indicates the number of proteins that are no longer expressed at all. [Diagram 3] Graph showing the number of proteins over- and under-expressed in HCT-116 MCB, DS-A and DS-B compared to primary HCT-116 RCB cultured in classical conditions (containing 10% FBS). [Figure 4] 1 is a graph showing the difference in the number of expressed proteins in HT-29 MCB, DS-A, DS-B, and the final product (DP) containing all six DSs (hence containing HT-29 DS-A and DS-B) compared to HT-29 RCB cultured in classical conditions (containing 10% FBS). "+" indicates newly expressed proteins and "-" indicates the number of proteins that are no longer expressed at all. [Diagram 5] Graph showing the number of over- and under-expressed proteins in HT-29 MCB, DS-A and DS-B compared to primary HT-29 RCB cultured in classical conditions (containing 10% FBS). [Figure 6] Graph showing the difference in the number of expressed proteins in LoVo MCB, DS-A, DS-B, and the final product (DP) containing all six DSs (hence containing LoVo DS-A and DS-B) compared to LoVo RCB cultured in classical conditions (containing 10% FBS). "+" indicates newly expressed proteins, and "-" indicates the number of proteins that are no longer expressed at all. [Figure 7] Graph showing the number of over- and under-expressed proteins in LoVo MCB, DS-A and DS-B compared to initial LoVo RCB cultured in classical conditions (containing 10% FBS). [Figure 8]HT-29 cells: HT-29 MCB, HT-29 DS-A, and HT-29 DS-B in several compositions, and in drug formulations (DPs) containing a mixture of HT-29 DS-A, HT-29 DS-B, HCT-116 DS-A, HCT-116 DS-B, LoVo DS-A, and LoVo DS-B, whether membrane and / or cell surface proteins or other (i.e., any other protein for which the term "membrane" or "cell surface" was not reported as an annotation in the "Cellular Component" region of the Thermo Protein Center database). Experiments were performed in triplicate and are indicated as "#1", "#2", and "#3". [Figure 9A] 9A-9D are pie charts showing protein distribution by p-value and fold change between HT-29 DS-A vs. HT-29 MCB (FIG. 9A), HT-29 DS-B vs. HT-29 MCB (FIG. 9B), HT-29 DS-A vs. HT-29 DS-B (FIG. 9C), and DP vs. HT-29 MCB (FIG. 9D). [Figure 9B] 9A-9D are pie charts showing protein distribution by p-value and fold change between HT-29 DS-A vs. HT-29 MCB (FIG. 9A), HT-29 DS-B vs. HT-29 MCB (FIG. 9B), HT-29 DS-A vs. HT-29 DS-B (FIG. 9C), and DP vs. HT-29 MCB (FIG. 9D). [Figure 9C] 9A-9D are pie charts showing protein distribution by p-value and fold change between HT-29 DS-A vs. HT-29 MCB (FIG. 9A), HT-29 DS-B vs. HT-29 MCB (FIG. 9B), HT-29 DS-A vs. HT-29 DS-B (FIG. 9C), and DP vs. HT-29 MCB (FIG. 9D). [Figure 9D] 9A-9D are pie charts showing protein distribution by p-value and fold change between HT-29 DS-A vs. HT-29 MCB (FIG. 9A), HT-29 DS-B vs. HT-29 MCB (FIG. 9B), HT-29 DS-A vs. HT-29 DS-B (FIG. 9C), and DP vs. HT-29 MCB (FIG. 9D). [Figure 10]Graph showing the number of membrane and / or cell surface proteins (on the y-axis) that are statistically significantly under-expressed (i.e., p-value≦0.05 and abundance ratio≦0.5) or over-expressed (i.e., p-value≦0.05 and abundance ratio≧2) between HT-29 DS-A vs. HT-29 MCB, HT-29 DS-B vs. HT-29 MCB, HT-29 DS-A vs. HT-29 DS-B, and DP vs. HT-29 MCB. The number of under-expressed proteins is shown as a negative number. [Figure 11A] Three graphs showing secretion of IL-8 or IL-12 from dendritic cells, expressed in pg / mL: Figure 11A: IL-8 secretion in the absence of CD40L; Figure 11B: IL-8 secretion in the presence of 0.6 μg / mL CD40L; Figure 11C: IL-12 secretion in the presence of 0.6 μg / mL CD40L. [Figure 11B] Three graphs showing secretion of IL-8 or IL-12 from dendritic cells, expressed in pg / mL: Figure 11A: IL-8 secretion in the absence of CD40L; Figure 11B: IL-8 secretion in the presence of 0.6 μg / mL CD40L; Figure 11C: IL-12 secretion in the presence of 0.6 μg / mL CD40L. [Figure 11C] Three graphs showing secretion of IL-8 or IL-12 from dendritic cells, expressed in pg / mL: Figure 11A: IL-8 secretion in the absence of CD40L; Figure 11B: IL-8 secretion in the presence of 0.6 μg / mL CD40L; Figure 11C: IL-12 secretion in the presence of 0.6 μg / mL CD40L. [Figure 12] 1 is a graph showing IFNγ release in supernatants collected from co-cultures of mDC and autologous CD8+ T cells treated with various ratios of STC-1010. The hashed line indicates the detection limit of IFNγ. [Figure 13A] Figure 13 shows three graphs showing expression of DC activation markers CD40, CD83 and CD86 assessed by RT-qPCR upon treatment with various doses of STC-1010. Data are expressed as relative amounts normalized to chicken GAPDH expression. Figure 13A: CD40 expression; Figure 13B: CD83 expression; Figure 13C: CD86 expression. [Figure 13B] Figure 13 shows three graphs showing expression of DC activation markers CD40, CD83 and CD86 assessed by RT-qPCR upon treatment with various doses of STC-1010. Data are expressed as relative amounts normalized to chicken GAPDH expression. Figure 13A: CD40 expression; Figure 13B: CD83 expression; Figure 13C: CD86 expression. [Figure 13C] Figure 13 shows three graphs showing expression of DC activation markers CD40, CD83 and CD86 assessed by RT-qPCR upon treatment with various doses of STC-1010. Data are expressed as relative amounts normalized to chicken GAPDH expression. Figure 13A: CD40 expression; Figure 13B: CD83 expression; Figure 13C: CD86 expression.

[0190] Working Example The present invention is further illustrated by the following examples. Example 1 Selection of cell lines for human colorectal cancer vaccines The inventors aimed to develop anti-cancer treatments that modulate cancer cell resistance mechanisms against themselves by artificially developing in vitro resistance mechanisms in cancer cell lines and rendering them immunogenic. Several colorectal human cell lines were selected as suitable candidates for the development of a human colorectal cancer vaccine.

[0191] The initial selection of cell lines was based on their biological and genetic characteristics to cover the largest clinical typology panel: microsatellite stability (MSS vs. MSI status); mutated vs. wild-type BRAF, KRAS, PIK3CA, Pten and TP53 genes; known resistance to common anti-cancer therapies (fluorouracil "5-FU", oxaliplatin, SN-38 (7-ethyl-10-hydroxycamptothecin), anti-VEGF antibodies). Based on the literature and databases (SelTARbase, Cellosaurus and GDSC databases), the following cell lines were selected: -LS174T (ATCC (registered trademark) reference number: CL-188 (trademark)), -SW620 (ATCC® Reference Number: CCL-227™), -SW480 (ATCC® Reference Number: CCL-228™), -LoVo (ATCC (registered trademark) reference number: CCL-229 (trademark)), -SW48 (ATCC® Reference Number: CCL-231™), - HCT-116 (ATCC Reference Number: CCL-247™), and -HT-29 (ATCC® Reference Number: HTB-38™).

[0192] Preliminary Research Cell Bank (RCB): These cell lines were obtained from ATCC and cultured in 10% FBS to obtain 7 RCBs, however, only 6 of these 7 had the expected growth profile and were viable after freeze-thaw cycles: SW620 was discarded at this stage (Table 1). [Table 1]

[0193] Pre-RCB 2%: These six RCBs with expected growth profiles were adapted to low serum culture conditions (10% FBS → 5% FBS → 2% FBS) to mimic the nutrient depletion effect observed with anti-VEGF antibodies. Only four cell lines were able to grow in low serum culture conditions and were viable after freeze-thaw cycles (HCT-116, HT-29, LoVo, and SW480) (Table 2). [Table 2] These four cell lines were then exposed to two additional types of stress, resulting in "DS-A" and "DS-B."

[0194] DS-A: To induce expression of the heat shock protein HSP70, cells were exposed to physical stresses including a combination of (1) low-dose ionizing radiation (10 Gy) for 5 min and (2) heat stress (42°C for 1 h).

[0195] DS-B: Cells were exposed to chemical stresses including chemical challenge with chemotherapeutic agents commonly used against colorectal cancer: 5-FU (Sigma, F6627), oxaliplatin (Sigma, Y0000271) or SN-38 (7-ethyl-10-hydroxycamptothecin) (Sigma, H0165). In particular, the association of chemotherapeutic agents with each cell line was based on literature precedents (Table 3) and further on the degree of resistance of the cell line to each chemotherapeutic agent. For each cell line, this level of resistance to each chemotherapeutic agent was measured as the 50% inhibitory concentration (IC) in a cytotoxicity assay. 50 The IC 50 is shown in Table 4. [Table 3] [Table 4]

[0196] After exposure to stress, whether physical or chemical, the cells became haptenized, i.e., immunogenic, through the attachment of a carrier molecule capable of conferring immunogenicity to stress proteins expressed in response to stress as a resistance mechanism, regardless of whether these stress proteins are free, bound to the surface of, or present within, the cancer cells. The carrier molecule is dinitrophenyl. Finally, three cell lines with the best growth potential in medium with reduced serum (2% FBS) after stress exposure were selected: LoVo, HCT-116 and HT-29.

[0197] Master Cell Bank (MCB): These three selected cell lines were cultured in low serum culture conditions (2% FBS) that mimicked metabolic stress (nutrient depletion observed during treatment with anti-VEGF antibodies) to obtain a Master Cell Bank (MCB).

[0198] Example 2 Human colorectal cancer vaccine manufacturing process Based on the preliminary results obtained in Example 1, the inventors prepared a vaccine composition comprising multiple stressed, haptenized, non-proliferating HT-29, HCT-116 and LoVo cells. For each cell line, DS-A and DS-B were produced starting from MCB. For DS-A, MCBs of each cell line were thawed (if frozen) and cultured in vitro. During their growth phase or their stationary phase, physical stress was applied, including low doses of ionizing radiation (10 Gy) for 5 min, along with heat stress (1 h at 42°C). Stress proteins expressed in response to this stress were then haptenized with dinitrophenyl. After formulation at 30 million cells / mL and freezing for storage purposes, these cells (containing the haptenized stress molecules) were inactivated, i.e., rendered non-proliferative, with high ionizing radiation (25 Gy), inhibiting cell proliferation while keeping the cellular structure intact. For DS-B, MCBs of each cell line were thawed (if frozen) and cultured in vitro. During their growth phase or their stationary phase, chemical stress was applied, including chemical stimulation with chemotherapeutic agents: for HT-29 cells, 13 μM oxaliplatin was applied for 72 h; for HCT-116 cells, 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) (or 100 nM SN-38 with similar end results in recent experiments) was applied for 48 h; and for LoVo cells, 5 μM 5-FU was applied for 48 h. The stress proteins expressed in response to this stress were then haptenized with dinitrophenyl. After formulation at 30 million cells / mL and freezing for storage purposes, these cells (containing the haptenized stress molecules) were inactivated, i.e., rendered non-proliferative, with high ionizing radiation (25 Gy), inhibiting cell proliferation while keeping the cellular structure intact.

[0199] A comparison of marker expression was performed to validate each DS-A and DS-B. For the three DS-As, radiation and heat stress applied to the three cell lines induced the same phenotypic changes in all three DS-As, overexpressing HSP70 and CD227 compared to non-stressed cells (Table 5). These data were confirmed in experiments performed with several other cell batches treated similarly, all confirming the overexpression of HSP70 and CD227 after radiation and heat stress. [Table 5]

[0200] In the case of DS-B, treatment of HT-29 cells with oxaliplatin resulted in overexpression of the CD95, CD107 and CD54 markers compared to non-stressed cells; and treatment of HCT-116 cells with SN-38 (7-ethyl-10-hydroxycamptothecin) or treatment of LoVo cells with 5-FU resulted in overexpression of the CD66 marker, and treatment of LoVo cells with 5-FU resulted in overexpression of CD243, compared to non-stressed cells (Table 6). These data were confirmed in experiments carried out with several other batches of cells treated similarly, all of which confirmed the following: -Overexpression of CD66 in HCT-116 cells after treatment with SN-38, regardless of the preliminary cell culture conditions (T25 or T225 CellStack); - overexpression of CD54, CD95 and CD107 in HT-29 cells after treatment with oxaliplatin; and -Overexpression of CD243 (and, to a lesser extent, CD66) in LoVo cells following treatment with 5-FU.

[0201] Finally, the vaccine composition (DP or "STC-1010") can be formulated by pooling all six DSs (three DS-A: one for each cell line; and three DS-B: one for each cell line) into a 100 μL dose (each containing 3 million cells). FACS analysis revealed high expression of HSP70, CD227, CD95 and CD243 in this final product (assessed as percentage of marker-expressing cells in total cell count and as mean fluorescence intensity) (Table 7). [Table 6] [Table 7]

[0202] Example 3 LC-MS / MS identification and relative quantification of proteins in six intermediate compositions During this study, various cell lines of different classifications, both with and without in vitro exposure to irradiation, heat stress, chemical stress, metabolic stress or combinations thereof, were analyzed. The expressed proteins were identified in specific databases. Adapted sample preparation was performed to improve protein detection. The individual amounts of the detected proteins were assessed. The normalized signal obtained for each protein was compared with the others.

[0203] material and method During production, cell lines should be harvested according to gene expression after exposure to different stresses (irradiation, heat, chemical, metabolic or combinations thereof). These different stresses induced overexpression of antigens. Immunogenicity was also enhanced by chemically labeling surface proteins with haptens. Protein expression was compared between different cell samples after exposure to different stresses. A summary of the different compositions analyzed in this study is shown in Table 8. [Table 8]

[0204] Sample preparation methods The sample preparation method applied to the cell pellets for protein detection involved chemical lysis of the cells followed by protein digestion. Each generated peptide was separated according to its physicochemical properties using a nanoflow chromatographic system coupled with a high-resolution mass spectrometer (nanoLC-MS / MS). The main advantage of using this technique is that it improves the sensitivity of the instrument and increases the number of proteins identified.

[0205] Analysis method NanoLC-MS / MS analysis was performed in data-dependent analysis (DDA) mode, also called shotgun proteomics or peptide mapping. This analytical tool allows the acquisition of MS and MS / MS spectra of thousands of peptides throughout the entire chromatographic separation.

[0206] Protein Identification Once experimental MS and MS / MS spectra were acquired, data processing was performed using a software search engine that uses mass spectrometry data to identify proteins from proteome databases, where the experimental data was correlated with the complete human proteome (SwissProt database) for protein identification.

[0207] To validate the identified proteins, several specific parameters were implemented to ensure specificity and eliminate false positives. Protein identification was performed with two peptides along with at least one protein-specific peptide to be very stringent with respect to the identified proteins. The identified proteins were those present in higher abundance. Furthermore, for the identification method, several frequently observed peptide modifications such as methionine oxidation or pyroglutamic acid formation from glutamine were added to the search engine. Protein abundance normalization was also performed.

[0208] Individual quantification evaluation This strategy involved adding a universal standard curve of synthetic peptides precisely calibrated by Readybeads™ technology (Anaquant, Villeurbanne, France). This standard curve allowed the conversion of peptide signals to the amounts of all specified proteins. The peptides used in the standard curve were selected for their specificity; their sequences did not correspond to any of the proteomes of the cell lines used in the bioprocess. These standards represent an accurate calibration curve that can be adapted to quantify the amount of any protein. The reproducibility and stability of this calibration curve is guaranteed by Readybeads™ technology. All identified peptides and proteins can be normalized using this calibration curve. The normalized signal allows for batch-to-batch comparisons.

[0209] result Thirteen samples were processed and analyzed simultaneously. Some pellet samples were difficult to dissolve, and protein quantification was performed prior to digestion using the Pierce™ BCA Protein Assay Kit to compare the same amount of protein injected. Despite this quantification step, after sample analysis, the amount of protein injected appeared to vary significantly between samples. The difference may result from differences in the amount of protein injected or from differences in the dynamic range of the protein in the samples. To evaluate these hypotheses, AQTBeads spiked into the samples were used as a quality control. Readybeads™ quality control was used to measure the gradient and r 2 Good linearity with a δ>0.9 was demonstrated as well as quantification of injected protein amounts ranging from 1 to 500 fmol. Peptide digests were also quantified after LC-MS injection. The results are shown in Table 9. [Table 9]

[0210] Whole protein comparison Untreated cells RCB (Research Cell Bank) samples were untreated samples in 10% FBS medium. The MCB (Master Cell Bank) was derived from the RCB that was medium adapted (2% FBS). The proteins identified in three human cell lines (HT-29, HCT-116, and LoVo) were compared (Figures 1A and 1B). Nearly 50% of the identified proteins were routinely identified in all three untreated human cell lines, a result expected since these three cell lines were derived from colon or colorectal cancer.

[0211] DS-A Treatment DS-A treatment involves exposing cells to a low dose of ionizing radiation (10 Gy) for 5 min along with heat stress (1 h at 42 °C). These cells were also subjected to metabolic stress (i.e., medium adaptation to 10% to 2% FBS during RCB and MCB). After this DS-A treatment, several proteins appeared to be overexpressed in all three cell lines (protein ratios between DS-A and MCB samples relative to total protein >2.5). These results are shown in Table 10. [Table 10] These 10 proteins, identified only in the DS-A samples but not in the MCB samples, reflect that DS-A treatment induces cells to develop resistance mechanisms, thereby producing stress proteins.

[0212] DS-B treatment DS-B treatment included exposure of cells to chemotherapeutic agents, i.e., 13 μM oxaliplatin for 72 h for HT-29 cells, 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for 48 h for HCT-116 cells, and 5 μM 5-FU for 48 h for LoVo cells. These cells were also subjected to metabolic stress (i.e., medium adaptation to 10% to 2% FBS during RCB and MCB). After this DS-B treatment, several proteins appeared to be overexpressed (protein ratios between DS-B and MCB samples relative to total protein amounts >2.5), and these proteins are reported in Table 11. [Table 11] The overexpressed proteins appeared to be mainly related to DNA repair.To analyze the results more deeply, we decided to focus on membrane proteins.

[0213] Focus on membrane proteins Untreated cells Tables 12-14 show the overexpressed membrane proteins (protein to total protein ratio >2.5) in one human cell line (untreated RCB and MCB) compared to the other two cell lines. [Table 12] [Table 13] [Table 14]

[0214] DS-A Treatment DS-A treatment involves exposure of cells to a low dose of ionizing radiation (10 Gy) for 5 min along with heat stress (1 h at 42 °C). These cells were also subjected to metabolic stress (i.e., medium adaptation to 10% to 2% FBS during RCB and MCB). Membrane proteins appeared to be primarily overexpressed rather than underexpressed. Tables 15-17 list the membrane proteins that were overexpressed (percentage ratio of protein to total protein >2.5) in each human cell line (DS-A sample) compared to its corresponding MCB sample. [Table 15] [Table 16] TIFF2024508003000017.tif61162 [Table 17]

[0215] DS-B treatment DS-B treatment included exposure of cells to chemotherapeutic agents, i.e., 13 μM oxaliplatin for 72 h for HT-29 cells, 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for 48 h for HCT-116 cells, and 5 μM 5-FU for 48 h for LoVo cells. These cells were also subjected to metabolic stress (i.e., medium adaptation to 10% to 2% FBS during RCB and MCB). Membrane proteins appeared to be primarily overexpressed rather than underexpressed. Tables 18-20 list the membrane proteins that were overexpressed (protein ratio >2.5 relative to total protein) in each human cell line (DS-B sample) compared to its corresponding MCB sample. [Table 18] [Table 19] [Table 20]

[0216] Example 4 LC-MS / MS identification and relative quantification of proteins in the final composition This study aimed to identify differentially expressed proteins in the final vaccine composition (DP, which contains all six of the above DS pooled together (3 DS-A: one of each cell line; and 3 DS-B: one of each cell line)). Figure 2 shows the difference in the number of proteins expressed in HCT-116 MCB, DS-A, DS-B, and the final product (DP) containing all six DSs (and thus HCT-116 DS-A and DS-B) compared to HCT-116 RCB cultured in classical conditions (containing 10% FBS). "+" indicates newly expressed proteins and "-" indicates the number of proteins that are no longer expressed at all. FIG. 3 shows the number of over- and under-expressed proteins in HCT-116 MCB, DS-A and DS-B compared to primary HCT-116 RCB cultured in classical conditions (containing 10% FBS). Figure 4 shows the difference in the number of expressed proteins in HT-29 MCB, DS-A, DS-B, and the final product (DP) containing all six DSs (thus containing HT-29 DS-A and DS-B) compared to HT-29 RCB cultured in classical conditions (containing 10% FBS). "+" indicates newly expressed proteins and "-" indicates the number of proteins that are no longer expressed at all. FIG. 5 shows the number of over- and under-expressed proteins in HT-29 MCB, DS-A and DS-B compared to primary HT-29 RCB cultured in classical conditions (containing 10% FBS). Figure 6 shows the difference in the number of expressed proteins in LoVo MCB, DS-A, DS-B, and the final product (DP) containing all six DSs (thus containing LoVo DS-A and DS-B) compared to LoVo RCB cultured in classical conditions (containing 10% FBS). "+" indicates newly expressed proteins. "-" indicates the number of proteins that are no longer expressed at all. FIG. 7 shows the number of proteins that were over- and under-expressed in LoVo MCB, DS-A and DS-B compared to primary LoVo RCB cultured in classical conditions (containing 10% FBS).

[0217] A comparison of these LC / MS data is shown in Table 21, which shows the number and percentage of proteins that are only present in the final composition (DP) (i.e., not identified in any of the three cell lines at the RCB stage), that are overexpressed in the final composition (DP) (compared to the three cell lines at the RCB stage), and that are similarly or less expressed in the final composition (DP). [Table 21] Table 22 shows a list of proteins that were found only in the final composition (DP) and not in any of the RCBs. The presence of any of these proteins is therefore characteristic of the final composition that underwent all the different treatments described above. [Table 22] Further analysis of the 1556 proteins identified in TIFF2024508003000024.tif239162 final composition (DP) showed that 97 of them were particularly important and could be classified into 12 superfamilies based on biological, clinical, and cancer prognostic value. Of the 97 proteins in the final composition (DP), 52 were overexpressed compared to the three cell lines at the RCB stage, and 8 were found only in the final composition (DP) but not in any of the RCBs. Table 23 summarizes the number of proteins in these 12 superfamilies. [Table 23] "Adhesion" corresponds to CAM proteins (cell adhesion molecules), including IgCAMs (eg, ICAM1), cadherins, integrins, and selectins. The "ATP-binding cassette" corresponds to a transmembrane protein of the transport system superfamily, which is involved in the phenomenon of drug resistance. "BCL" refers to proteins that regulate cell death, either pro-apoptotic (e.g., BAX, BAK1 / Bcl-2 cognate antagonist killer, and Bcl-2-associated death promoter) or anti-apoptotic (e.g., Bcl-2, and Bcl-xL). "COX" corresponds to the cytochrome C oxidase protein (also called "complex IV"), a protein from the terminal component of the mitochondrial respiratory chain. Mutations in cytochrome C oxidase are implicated in cancer (especially mutations in cytochrome C oxidase subunit 4). "EGFR" corresponds to epidermal growth factor, which is involved in the pathogenesis and progression of various carcinoma types. "HSPs" correspond to heat shock proteins, a class of proteins that are overexpressed in a wide range of human cancers and are involved in tumor cell proliferation, differentiation, invasion, metastasis, death, and recognition by the immune system. "Inhibitors" correspond to proteins that are associated with promoting or anti-cancer proliferation. "MUC" corresponds to mucin protein, a highly glycosylated protein. MUC13 is frequently and aberrantly expressed in various epithelial cancers, including gastric, colorectal, and ovarian cancers. "RAS-related" refers to Rap GTP-binding proteins, a type of small GTPase. More than 30% of all human cancers are driven by mutations in the RAS family of genes, including 95% of pancreatic cancers and 45% of colorectal cancers. "Repair" corresponds to a protein associated with tumor progression. "Transporters" correspond to transmembrane proteins that have a function in drug resistance. Table 24 identifies these 52 proteins that are overexpressed, 8 of which are only expressed in the final composition (DP) compared to RCB. [Table 24] TIFF2024508003000027.tif237162TIFF2024508003000028.tif242161 Table 25: shows the relationship between the biological, clinical and cancer prognostic values ​​of these 97 proteins in various types of cancer.As can be seen, the final composition (DP) expresses markers that are not only related to colorectal cancer, as expected considering that HT-29, HCT-116 and LoVo cells are colorectal cell lines, but also related to other types of cancer.This suggests that the compositions described herein may be useful not only for the treatment of colorectal cancer, but also for the treatment of a wide variety of other cancers. [Table 25]

[0218] Example 5 Identification and relative quantification of surface proteins in two intermediate compositions and the final product by LC-MS / MS This study aimed to characterize the surface proteome of neoplastic cells at different production steps in one starting material (HT-29 MCB), two corresponding intermediate compositions (HT-29 DS-A and HT-29 DS-B) and the final product (DP) containing all six of the above (hence containing HT-29 DS-A and HT-29 DS-B) and finally to identify differentially expressed surface proteins.

[0219] material and method Similar types of products (DS / DP) were analyzed as described in previous examples 3 and 4. The same steps were applied, in particular exposure to different stresses (irradiation, heat, chemical, metabolic or combinations thereof). These different stresses induced overexpression of antigens. Immunogenicity was also enhanced by chemically labeling surface proteins with haptens. Protein expression was compared between different cell samples after exposure to different stresses. A summary of the different compositions analyzed in this study is shown in Table 26. [Table 26]

[0220] Sample processing 6x10 when possible 6 A quantity of cells (if not possible, the entire sample was used) was gently washed with PBS and then biotinylated using Pierce™ Cell Surface Protein Biotinylation. Cells were then lysed and proteins were isolated using the Pierce™ Cell Surface Protein Biotinylation and Isolation Kit (Thermo Scientific, Cat. No. A44390). All preparation steps were performed according to the manufacturer's instructions. Proteins were then precipitated with methanol-chloroform, the precipitate washed with methanol, dried and solubilized by microcavitation (Bioruptor Pico, Diagenode) in iST LYSE (PreOmics Gmbh) buffer. Proteins were digested with LysC and trypsin. Peptides were purified using a mixed-mode reversed-phase cation exchanger SPE column (PreOmics Gmbh) and, after drying, solubilized in 100 μL of 3% acetonitrile, 0.1% formic acid in water. Peptide concentrations were measured using the BCA method (Table 27). Although the measured peptide concentrations were low, their amounts were sufficient for this feasibility study. [Table 27]

[0221] Analysis method LC-MS / MS 250 ng of peptide was injected in triplicate for each sample. Chromatography was performed using an Ultimate 3000 (Dionex) instrument equipped with a RepMap100 C18 (75 μm×50 cm, 2 μm material) column, applying a 120 min gradient of 2.5% to 35% acetonitrile at a flow rate of 300 nL / min after a 3 min trapping step on the precolumn. Data were acquired using a Q-Exactive (Thermo) mass spectrometer using the experimental settings described in Table 28. MS / MS scans were performed on the 10 most intense ions of each cycle, resulting in 6545 cycles, thus an average of 17 cycles per chromatographic peak. [Table 28]

[0222] Protein Identification Data were processed with Proteome Discoverer 2.4. Proteins were identified using the SEQUEST-HT algorithm against a database collecting the human reference proteome obtained from NeXtProt and the cRAP contaminant database excluding human proteins. The parameters were as follows: -enzyme = trypsin(complete); -Tolerated mis-disconnects=2; -Precursor error tolerance = 10 ppm; -Fragment error tolerance=0.02Da; -Dynamic modifications = oxidation (M), deamidation (N / Q), CAM thiopropanoyl (K); -Protein terminal modifications = acetylation, CAM thiopropanoyl; -Static modification = carbamidomethyl (C). False discovery rate (FDR) measurements were performed using the Percolator algorithm. All spectra reported with less confidence than SEQUEST-HT, i.e. considered unidentified, were processed a second time with the same algorithm against the same database as above, but with the following modified settings: -enzyme = trypsin_R(half); -Tolerated mis-disconnects=2; -Precursor error tolerance = 10 ppm; -Fragment error tolerance=0.02Da; -Dynamic modifications = oxidation (M), deamidation (N / Q), CAM thiopropanoyl (K); -Protein terminal modifications = acetylation, CAM thiopropanoyl; -Static modification = carbamidomethyl (C). False discovery rate (FDR) measurements were performed using the Percolator algorithm. A protein was considered to be part of the "membrane" or "cell surface" if the keywords "membrane" or "cell surface" were reported as annotations in the "cellular component" region of the Thermo Protein Center database. It should be noted that several cellular components may be reported for the same protein. Furthermore, the term "membrane" may refer to membranes other than the cell membrane (e.g., nuclear membrane, organelle membrane, vesicle membrane, etc.).

[0223] Protein quantification Data were processed using Minora and feature mapper for Proteome Discoverer 2.4 software. The peak integration parameters were as follows: -Post-acquisition recalibration=Yes (precision parameters); -min trace length=5; -Maximum delta RT for isotope = 0.2 min; -PSM confidence level of integral = high. The chromatographic alignment parameters were as follows: -RTAlign=Yes: -parameter tuning = precision; -Maximum RT shift = 5 min; -Mass tolerance = 10 ppm. The feature mapping parameters were as follows: -RTTolerance=Auto; -mass tolerance=auto; -S / N threshold=2. Statistical analysis was performed by using the Precursor Ion Quantifier node for Proteome Discoverer 2.4 software. The general quantification settings were as follows: -Peptides used = UNIQUE + RAZOR (UNIQUE means peptides that are not shared by different proteins or protein groups; RAZOR means peptides that are shared by multiple protein groups but are only used to quantify the protein with the largest number of unique peptides and the longest amino acid sequence); -Consider protein groups for peptide uniqueness = Yes; -Reject quantitative analysis results with missing channels = None; Precursor quantification settings were as follows: -Precursor abundance = area based; -Minimum number of recurring features = 50% (a peptide must be detected in at least 50% of a group of samples to be used for quantification). The normalization settings were: -Normalization mode = total peptide abundance (the sum of the abundance values ​​of each injection for all peptides identified is calculated and the injection with the highest total abundance is used as the reference to correct the abundance values ​​of all other injections by a constant factor per injection, so that the final total abundance is the same for all injections). The quantitative analysis roll-up hypothesis testing setup was as follows: - Ratio calculation = pairwise ratio based (peptide ratios are calculated as the geometric median of all combination ratios from all replicates for the selected test factor. Protein ratios are then calculated as the geometric median of peptide group ratios). - imputation mode = replicate-based resampling (replace missing values ​​with random values ​​sampled from a distribution centered around the median of the detected values ​​of the (technical and biological) replicates); -Hypothesis testing = t-test (background based). The hypothesis test that gives a p-value is the t-background test (or background ANOVA). This test is based on the assumption that most protein abundances do not change in response to stimuli in proteomics. This method determines the rank of protein ratios that are considered to be mostly constant between conditions before testing the abundance ratios of each protein against the median and variance of this constant population. This test is useful for tests with missing values ​​and can be used only when several hundred proteins are identified. It does not require technical replicates.

[0224] result Protein detection count The number of proteins and peptides identified in each sample are shown in Table 29 and FIG. [Table 29]

[0225] Relative protein quantification Four comparisons were performed comparing: -HT-29 DS-A and HT-29 DS-B, -HT-29 DS-A and HT-29 MCB, -HT-29 DS-B and HT-29 MCB, and -HT-29 MCB and DP. Note 1: Some protein abundance values ​​may be missed due to biological or technical variability and / or the stochastic nature of mass spectrometry acquisition of trace data. Therefore, we consider a protein quantifiable if its quantifiable peptide is present in at least 50% of the infusions in any group. Note 2: For some proteins or peptides, the abundance ratios are equal to 1000 or to 0.001. These values ​​are arbitrary. In the first case, they mean that this protein or peptide is quantified only in molecular terms, in the second case, only in denominator terms. Note 3: Proteins that are not identified based on the MS / MS fragmentation spectra of their peptides can still be quantified by "match between runs", based on the similarity of their XIC(MS1) chromatographic properties (accurate mass, retention time) to those of peptides identified in at least one acquisition of the same experiment. Note 4: Protein abundances are considered statistically and significantly different if they have an abundance ratio of 0.5 or less or 2 or more, with an associated p-value of 0.05 or less. Figures 9A-D show the distribution of proteins according to p-value and fold change in each of the four comparisons. FIG. 10 shows the number of membrane and / or cell surface derived proteins with statistically significant differences in each of the four comparisons. For all injections, 65% of the identified proteins were associated with the terms "membrane" or "cell surface" in the "Cellular Components" region of the Thermo Protein Center database (Figure 8). In comparison, in non-biotinylated HeLa cells, a lower proportion of identified proteins (51%, i.e., 816 of 3562) were associated with the terms "membrane" or "cell surface" in the same database. This tends to indicate successful enrichment of the surface proteome in all four samples. Relative quantification and comparison between samples showed that approximately 29%-34% of proteins were significantly altered when comparing stressed / haptenized cells (DS-A or DS-B) or drug formulations (DP) with untreated cells (MCB) (Figure 9A-C, respectively). A lower percentage (15%) is observed when comparing both stressed / haptenized cell samples (DS-A and DS-B) with each other (Figure 9D). More than half of the significantly over- or under-expressed proteins are annotated as membrane and cell surface proteins (FIG. 10).

[0226] Comparison results HT-29 DS-A / HT-29 MCB Based on the raw data, an abundance ratio DS-A / MCB > 1000 was chosen to select membrane or cell surface proteins that were overexpressed after haptenization following radiation and heat shock stress. When considering cell membrane proteins, 455 proteins with abundances > 1000 were identified as overexpressed in DS-A compared to MCB. When considering specific cell surface proteins, 127 proteins with abundance ratios > 2 were identified as overexpressed in DS-A compared to MCB, of which 38 had abundance ratios of 1000 (Table 30). [Table 30] HT-29 DS-A / HT-29 MCB Based on the raw data, an abundance ratio DS-A / MCB > 1000 has been chosen to select membrane or cell surface proteins that are overexpressed after heat shock followed by haptenization. When considering plasma membrane proteins, 430 proteins with abundances > 1000 were identified as overexpressed in DS-B compared to MCB. When considering specific cell surface proteins, 127 proteins with abundance ratios > 2 were identified as overexpressed in DS-B compared to MCB, of which 37 had abundance ratios of 1000 (Table 31). [Table 31] DP / HT-29 MCB NOTE: Due to the composition of the DP collecting HT-29 DS-A and HT-29 DS-B as well as HCT-116 DS-A, HCT-116 DS-B, LoVo DS-A and LoVo DS-B, this comparison is not representative of actual protein overexpression; therefore, for MCB comparisons it will be necessary to take dilution factors into account. Based on the raw data, an abundance ratio DP / MCB > 1000 is chosen to select overexpressed membrane or cell surface proteins. When considering plasma membrane proteins, 343 proteins with abundances > 1000 were identified as overexpressed in the DP compared to the MCB. When considering specific cell surface proteins, 112 proteins with abundance ratios > 2 were identified as overexpressed in the DP compared to the MCB, of which 34 had abundance ratios of 1000 (Table 32). [Table 32]

[0227] Proteome characterization in drug formulations (DP) Table 33 lists the 132 cell surface proteins identified in three replicates of a DP containing a mixture of HT-29 DS-A, HT-29 DS-B, HCT-116 DS-A, HCT-116 DS-B, LoVo DS-A and LoVo DS-B. [Table 33] TIFF2024508003000038.tif241160TIFF2024508003000039.tif242160TIFF2024508003000040.tif241162TIFF2024508003000041.tif38162

[0228] Example 6 In vitro assessment of the functional activity of STC-1010 by mixed lymphocyte reaction assay The objective was to evaluate the final product (DP or "STC-1010") for its ability to induce an immunogenic profile of human monocyte-derived dendritic cells (DCs) in vitro when applied alone or in combination with CD40L (naturally present in vivo) and to evaluate dendritic cell-mediated T cell activation in a mixed lymphocyte reaction (MLR) assay. The study was performed using HLA-matched monocyte-derived mDCs and CD8 + Co-cultures of T cells were performed in a system in which i) the cytokine profile of DCs was measured by quantification of cytokines released in the culture supernatant (including IL-12 and IL-8) and ii) DC-mediated T cell activation was measured by evaluation of the MLR response by quantification of released IFNγ using a specific homogeneous time-resolved fluorescence (HTRF)-based detection kit.

[0229] material and method i) monocytes, which are used to obtain mature DCs through a differentiation / maturation protocol, and ii) CD8 + To freshly isolate lymphocytes, human PBMCs from healthy donors were used in this study. Briefly, human monocytes were freshly isolated from PBMCs, cultured in the presence of GM-CSF and IL-4, and differentiated into DCs. At the end of the differentiation process, DCs were matured in a specific cocktail in the presence of LPS and IFNγ with STC-1010 at three different ratios (1:1, 3:1, and 10:1, plus no STC-1010 as a negative control), with and without 0.6 μg / mL CD40L. Upon maturation, mDCs express CD209 + CD1a + CD80 + CD83 + CD86 + As such, cell culture supernatants were harvested for quantification of IL-12 and IL-8 by HTRF. DCs were then incubated with CD8 +The control condition (no treatment), as well as T cells alone and mDC alone were included in the experiment. After 72 hours of co-culture, the supernatants were collected and analyzed for CD8 + The effect of STC-1010 on T cell activation was assessed by quantification of released IFNγ levels, which is used as an important representative surrogate of T cell activation. Cytokine level quantification was performed by HTRF.

[0230] result Effect of STC-1010 on mDC cytokines STC-1010 was able to limit IL-8 secretion by mDCs in a ratio-dependent manner in both the presence and absence of CD40L (FIGS. 11A and 11B, respectively). STC-1010 was also able to enhance IL-12 secretion by mDCs in the presence of CD40L in a ratio-dependent manner (FIG. 11C). This ratio-dependent response confirms the effect of STC-1010 on DC maturation.

[0231] Two other cytokines (IL-10 and TNFα) were also evaluated during the course of this study: STC-1010 demonstrated a tendency to limit IL-10 secretion in the presence of CD40L; whereas TNFα secretion was slightly reduced by STC-1010 exposure in both the absence or presence of CD40L, with the effect maximal at the highest ratio. Effect of STC-1010 on MLR responses by IFNγ quantification Home CD8 + mDCs co-cultured with T cells, when previously exposed to CD40L during maturation, demonstrated effective MLR responses, as evidenced by the significant upregulation of IFNγ release in supernatants collected from the co-cultures compared to control and vehicle ( FIG. 12 ).

[0232] conclusion Considering these data, STC-1010 demonstrated its ability to modulate DC maturation along with its immunogenicity and rate-dependent activity as evidenced through cytokine profiles. This immunogenic profile was confirmed by MLR and was consistent with that observed in CD8 + The T cells had improved functional activity.

[0233] Example 7 In ovo assessment of functional activity of STC-1010 by chorioallantoic membrane assay The objective was to evaluate the final product (DP or "STC-1010") in ovo for its ability to induce an immune response in a chorioallantoic membrane (CAM) assay.

[0234] material and method Preparation of chicken embryos Fertilized White Leghorn eggs were incubated at 37.5°C and 50% relative humidity for 9 days. At that time (E9), the CAM was dropped into the air sac by puncturing a small hole in the eggshell and a 1 cm drop was placed in the eggshell on top of the CAM. 2 At least 20 eggs were opened for each test group (however, because eggshell opening is an invasive surgical procedure and some mortality may occur during the first few hours after opening, data may be collected on 15-20 eggs per group).

[0235] treatment Prior to treatment, the viability of each egg was checked and the surviving eggs were randomized into groups. All groups of eggs were treated with a volume of 100 μL of STC-1010 under the following three test conditions: -Test condition "STC1010[1]"; 10 5 cells / mL, i.e., 10 4 cells / embryos; -Test condition "STC1010[2]"; 5x10 5 cells / mL, i.e., 5x10 4 Cells / embryos; and -Test condition "STC1010[3]"; 10 6 cells / mL, i.e., 10 5 cells / embryos. A negative control without STC-1010 ("Neg Ctrl") was run simultaneously. Quantitative assessment of chicken immune cells On day E18, peripheral blood was collected and treated with heparin to prevent blood clotting. For dendritic cell (DC) evaluation, 100 μL of individual samples (n=8 / group) were collected from which RNA was extracted, reverse transcribed, pre-amplified, and analyzed by qPCR using primers specific for chicken CD40, CD83, and CD86 sequences. For all points performed by qPCR, chicken GAPDH expression was also analyzed as a reference gene expression and was used to normalize immune biomarker expression between samples. The C of each sample was q Calculation of the average C q And the relative amounts of immune cells in each group were directly controlled by Bio-Rad® CFX Maestro software. The remaining blood samples were pooled within the groups for T-lymphocyte evaluation. Blood samples were then processed by Hypaque-Ficoll (HF) separation for peripheral blood mononuclear cells (PBMC) isolation. Purified PBMC were then labeled with anti-chicken CD45 (ThermoFisher, ref. MA5-28679), anti-chicken CD3 (Southern Biotech, ref. 8200-26), anti-chicken CD4 (Thermo Fisher, ref. MA5-28686) and anti-chicken CD8 (Thermo Fisher, ref. MA5-28686) for T-lymphocyte evaluation by flow cytometry analysis. Quantitative assessment of immune cytokines On day E18, peripheral blood was individually collected (n=5 / group) and treated with heparin to prevent blood clotting. Blood samples were then centrifuged for plasma collection, followed by ELISA analysis for IL-12 and IFNγ expression, with each plasma sample evaluated at three dilutions. All ELISA kits were ordered from Cusabio (Chicken IL-12 ELISA kit, reference number: CSB-E12836C; Chicken IFN ELISA kit, reference number: CSB-E08550Ch).

[0236] Statistical analysis and significance For all quantitative data, outlier identification and one-way ANOVA (post-hoc tests between each duplicate group) were performed using Prism® (GraphPad Software).

[0237] result Quantitative assessment of chicken immune cells by FACS CD45 in purified PBMCs at E18 + , CD3 + , CD4 + , and CD8 + Leukocyte activation in the groups "Neg Ctrl" and "STC-1010[3]" was evaluated by FACS quantification of cells. Table 34 shows the FACS analysis data of these different cell subsets contained in peripheral leukocytes (peripheral CD45 + (as % of white blood cells). [Table 34] These data are consistent with those of CD4 + and CD8 + Increase in white blood cells, especially CD3 - / CD4 + Shows a high increase in white blood cells.

[0238] Quantitative assessment of immune cytokines The expression levels of IL-12 and IFNγ in peripheral blood in all groups at E18 were estimated. To properly evaluate cytokine secretion, each plasma sample was evaluated at three dilutions: 1 / 2, 1 / 10 and 1 / 50. [Table 35] As can be seen from these results, a net increase in STC-1010-induced IL-12 secretion was observed in all three sample dilutions compared to the negative control, however, no significant increase in IFNγ was observed following STC-1010 treatment (data not shown).

[0239] Quantitative assessment of chicken immune cells by RT-qPCR Expression of three DC activation markers (CD40, CD83, CD86) was assessed by RT-qPCR. Results show STC-1010-induced upregulation of these markers, particularly CD40 and CD86 at the highest dose, and CD83 at all doses, with a more significant upregulation at the lowest dose (Figure 13A-C).

[0240] conclusion Throughout the study, the majority of embryos survived, confirming the lack of toxicity of STC-1010: -Negative control: n=50, 3 embryos died (6% mortality); - Group “STC1010[1]”: n=48, 2 embryos died (4.17% mortality); - Group “STC1010[2]”: n=33, 0 embryos died (0% mortality); - Group “STC1010[3]”: n=29, 1 embryo died (3.45% mortality). Using this CAM assay, STC-1010 was shown to be capable of enhancing the immune system and activating innate and / or adaptive immune responses. Based on these encouraging results, a new study will be conducted to evaluate the in ovo efficacy of STC-1010 on chicken embryos grafted with human colorectal adenocarcinoma.

Claims

1. (i) stressed HT-29, HCT-116 and LoVo cells, and (ii) compositions comprising immunogenic stress proteins produced by these cells in response to stress applied in vitro.

2. 2. The composition of claim 1, wherein stressed HT-29, HCT-116 and LoVo cells have developed resistance mechanisms in response to one or more stresses applied in vitro selected from the group consisting of irradiation, heat stress, chemical stress, metabolic stress and any combination thereof, which result in said production of stress proteins.

3. The composition of claim 1 or 2, wherein the stressed HT-29, HCT-116 and LoVo cells are non-proliferative.

4. The composition of any one of claims 1 to 3, wherein the immunogenic stress protein is haptenized.

5. The immunogenic stress protein is selected from the group consisting of 2,4-dinitrophenyl (DNP); 2,4-dinitrofluorobenzene; sulfanilic acid; N-iodoacetyl-N'-(5-sulfonic acid-naphthyl)ethylenediamine; aniline; p-aminobenzoic acid; biotin; fluorescein and derivatives thereof (including FITC, TAMRA, and Texas Red); digoxigenin; 5-nitro-3-pyrazolecarbamide; 4,5-dimethoxy-2-nitrocinamide; 2-(3,4-dimethoxyphenyl)-quinoline-4-carbamide; 2,1,3-Benzoxadiazole-5-carbamide; 3-hydroxy-2-quinoxalinecarbamide; 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL); Rotenone isoxazoline I(E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenodi)acetamide; The composition of any one of claims 1 to 4, which is haptenized with a hapten selected from the group consisting of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid; 2-acetamido-4-methyl-5-thiazolesulfonamide; and p-methoxyphenylpyrazopodophilamide.

6. The composition according to any one of claims 1 to 5, which is a pharmaceutical or vaccine composition and further comprises at least one pharma- ceutically acceptable excipient.

7. about 10 ５ ~about 10 ８ The composition of any one of claims 1 to 6, comprising HT-29, HCT-116 and LoVo cells subjected to individual stresses.

8. A composition according to any one of claims 1 to 7 for use in the treatment of cancer in a subject in need thereof.

9. Use of a composition described in any one of claims 1 to 7 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

10. An intermediate composition comprising: (i) one of stressed HT-29 cells, stressed HCT-116 cells, and stressed LoVo cells; and (ii) a stress protein, An intermediate composition, wherein said one of stressed HT-29 cells, stressed HCT-116 cells, or stressed LoVo cells have developed resistance mechanisms in response to in vitro applied (i) metabolic stress, (ii) radiation, and (iii) heat stress, which results in the production of said stress protein.

11. An intermediate composition comprising: (i) one of stressed HT-29 cells, stressed HCT-116 cells, or stressed LoVo cells; and (ii) a stress protein, An intermediate composition, wherein said one of stressed HT-29 cells, stressed HCT-116 cells, or stressed LoVo cells have developed resistance mechanisms in response to in vitro applied (i) metabolic stress and (ii) chemical stress, which results in the production of said stress protein.

12. A method for producing the intermediate composition according to claim 10 or 11, comprising the steps of: a) culturing HT-29, HCT-116 or LoVo cells in a suitable medium; b) subjecting the HT-29, HCT-116 or LoVo cells cultured in step a) to one or more stresses in vitro, whereby these HT-29, HCT-116 or LoVo cells develop resistance mechanisms in response to said one or several stresses, thereby producing stress proteins; c) recovering the stressed HT-29, HCT-116 or LoVo cells together with the stress proteins they produced in step b); d) treating the stressed HT-29, HCT-116 or LoVo cells, together collected in step c), and the stress proteins they produce with a molecule or method capable of rendering said stress proteins immunogenic; A method comprising:

13. 13. The method of claim 12, wherein step c) is carried out at least several hours after completion of step b).

14. The method of claim 12 or 13, wherein step c) is carried out at least 12 hours or later after completion of step b).

15. 15. The method of any one of claims 12 to 14, wherein step d) comprises linking or complexing the stress protein to a means capable of conferring immunogenicity.

16. The method of claim 15, wherein the means capable of conferring immunogenicity is a hapten.

17. The method of claim 1, wherein the hapten is 2,4-dinitrophenyl (DNP); 2,4-dinitrofluorobenzene; sulfanilic acid; N-iodoacetyl-N'-(5-sulfonic acid-naphthyl)ethylenediamine; aniline; p-aminobenzoic acid; biotin; fluorescein and derivatives thereof (including FITC, TAMRA, and Texas Red); digoxigenin; 5-nitro-3-pyrazolecarbamide; 4,5-dimethoxy-2-nitrocinamide; 2-(3,4-dimethoxyphenyl)-quinoline-4-carbamide; 2,1,3-Benzoxadiazole-5-carbamide; 3-hydroxy-2-quinoxalinecarbamide; 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL); Rotenone isoxazoline; (E)-2-(2-(2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)phenodi)acetamide; 17. The method of claim 16, wherein the compound is selected from the group consisting of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid; 2-acetamido-4-methyl-5-thiazolesulfonamide; and p-methoxyphenylpyrazopodophilamide.

18. Step b) comprises subjecting the HT-29, HCT-116 or LoVo cells cultured in step a) to the following stresses applied in vitro simultaneously or sequentially: (i) in vitro culture in depleted medium, under low oxygen, and / or at low pH; (ii) in vitro irradiation at a total dose ranging from about 0.25 to about 25 Gy; and (iii) an in vitro heat shock applied to the cells at a temperature ranging from about 38° C. to about 45° C. for a time ranging from about 15 minutes to about 4 hours; 18. A method according to any one of claims 12 to 17 for producing the intermediate composition according to claim 10, comprising subjecting

19. Step b) comprises simultaneously or sequentially applying in vitro to the HT-29, HCT-116 or LoVo cells cultured in step a) the following stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation with a total dose ranging from about 1 to about 15 Gy for a time ranging from about 1 to about 20 minutes; and (iii) in vitro heat shock at a temperature of about 42° C. for about 60 minutes; A method according to any one of claims 12 to 18 for producing the intermediate composition according to claim 10, comprising subjecting

20. Step b) comprises simultaneously or sequentially applying in vitro to the HT-29, HCT-116 or LoVo cells cultured in step a) the following stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro irradiation for about 1 to about 5 minutes with a total dose of about 10 Gy; and (iii) in vitro heat shock at a temperature of about 42° C. for about 60 minutes; 20. A method according to any one of claims 12 to 19 for producing the intermediate composition according to claim 10, comprising subjecting

21. Step b) comprises subjecting the HT-29, HCT-116 or LoVo cells cultured in step a) to the following stresses applied in vitro simultaneously or sequentially: (i) in vitro culture in depleted medium, under low oxygen, and / or at low pH; (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a time period ranging from about 6 hours to about 120 hours; A method according to any one of claims 12 to 17 for the preparation of an intermediate composition according to claim 11, comprising subjecting

22. Step b) comprises simultaneously or sequentially applying in vitro to the HT-29, HCT-116 or LoVo cells cultured in step a) the following stresses: (i) in vitro culture in low serum culture conditions in 2% FBS medium; (ii) in vitro exposure to at least one or more chemotherapeutic agents and / or alcohol for a time period ranging from about 6 hours to about 120 hours; A method according to any one of claims 12 to 17 for the preparation of an intermediate composition according to claim 11, comprising subjecting 23. The cell is an HT-29 cell, and the in vitro exposure in (ii) is exposure to about 13 μM oxaliplatin for about 72 hours; or the cells are HCT-116 cells and the in vitro exposure in (ii) is exposure to about 315 nM SN-38 (7-ethyl-10-hydroxycamptothecin) for about 48 hours; or the cells are LoVo cells, and the in vitro exposure in (ii) is exposure to about 5 μM fluorouracil (5-FU) for about 48 hours; 23. The method of claim 21 or 22.

24. a. Obtaining the six intermediate compositions of claims 10 and 11, The six intermediate compositions are: 1) the intermediate composition of claim 10, comprising stressed HT-29 cells and stress proteins; 2) the intermediate composition of claim 10, comprising stressed HCT-116 cells and stress proteins; 3) the intermediate composition according to claim 10, comprising stressed LoVo cells and stress proteins; 4) the intermediate composition of claim 11, comprising stressed HT-29 cells and stress proteins; 5) The intermediate composition of claim 11, comprising stressed HCT-116 cells and stress proteins; 6) the intermediate composition according to claim 11, comprising stressed LoVo cells and stress proteins; b. mixing these six intermediate compositions together; A method for producing the composition according to any one of claims 1 to 7, comprising:

25. The method of claim 24, wherein the six intermediate compositions are mixed together in equal proportions with stressed HT-29, HCT-116 and LoVo cells.

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