Compound for tissue conservation

Abstract

The invention relates to a method for preservation of a tissue or an organ previously obtained from a mammal, for transplant of said tissue or organ in a recipient compatible mammal, the method comprising: - contacting, preferably in vitro or ex vivo, said tissue or organ with a treating composition, the treating composition comprising a neddylation pathway inhibitor, for 1 to 12h hours, in order to obtain a treated tissue or organ, said composition comprising from 1 to 100 µM of said neddylation pathway inhibitor; - washing the treated tissue or organ with a conservation composition for removing the treating composition from the treated tissue or organ, in order to obtain a washed tissue or organ; and - contacting washed tissue or organ with a preservation composition, in order to obtain a ready to be transplanted tissue or organ.

Description

Compound for tissue conservation

[0001] The invention relates to a compound for its use for tissue conservation.

[0002] The isolation of an organ from the systemic circulation, storage, and transplantation are responsive of numerous changes in the homeostasis and metabolism of the organ. Alterations of homeostasis and metabolism result in damages leading to the deterioration of the organ, its later incompatibility with the recipient or delayed graft function.

[0003] Various protocols are used today to ensure that grafts remain as viable as possible between removal from the donor and implantation in the recipient. These are adapted according to the type of donor and may vary from country to country. For kidneys, for example, the preservation protocols currently used in France are as follows:

[0004] - For brain-dead donors with standard criteria (‘good quality’ grafts), the organs are simply stored statically in a preservation solution at 4°C in a sterile container. This simple and inexpensive procedure also makes it easier to transport the organs from the donor site to the transplant site.

[0005] - For donors in a state of brain death with extended criteria (‘lower quality’ grafts), the organs are mainly preserved on a hypothermic perfusion machine with a suitable perfusion solution, a method that has shown beneficial effects on the rates of delayed recovery of graft function.

[0006] - For donors who have died after controlled cardiac arrest, known as ‘Maastricht III’ donors, a normothermic regional perfusion is first established before the donor is harvested, and then the organs are preserved on a hypothermic perfusion machine.

[0007] The other solid organs (pancreas, heart, lung, liver) are currently mainly preserved statically in a preservation solution at 4°C, but dynamic perfusion techniques are expanding rapidly for all organs (clinical trials planned or underway for all organs, deployment of hypothermic and normothermic perfusion systems in liver transplantation).

[0008] These different methods make it possible to preserve the viability of the grafts according to their initial quality, and are all based on a method of ex vivo preservation in hypothermia, which makes it possible to significantly reduce cellular metabolism and oxygen requirements, and to maintain the quality of the grafts as they were at the time of removal. However, they all include a phase of cold ischemia and hypoxia that will cause damage to the organs. Despite the beneficial effect of hypothermic perfusion on delayed recovery of graft function, this method has no ‘conditioning’ effect and does not clearly improve organ quality or long-term graft survival.

[0009] Other preservation methods have been evaluated, including normothermic machine perfusion, to greatly reduce or eliminate the cold ischemia phase. This technique requires suitable perfusion solutions containing an oxygen carrier. However, keeping organs for too long under normothermic perfusion poses problems in terms of eliminating toxic products derived from cellular activity. In addition, this procedure is costly, requires a lot of human resources and is still difficult to implement on a routine basis.

[0010] Some preservation fluids are used for the preservation of pancreas, heart, kidney and lung with comparable effects. To prolong viability of the organ, oxygen and energy demands are to be kept at a minimum. Hypothermic storage is currently the primary means for preserving viability. Cooling itself, however, has detrimental effects on the tissue, due to e.g. oxidative stress and inflammation. Cold ischemia (or cold storage) time is an independent risk factor of delayed graft function and primary organ. It has also been described that human cadaveric renal transplants have significantly more apoptotic cells than living-related transplants. The degree of apoptosis correlated significantly with the duration of cold ischemia.

[0011] Perfusion storage reduces apoptosis in a porcine kidney model of donation after cardiac death. It has been shown by several authors that organs from extended criteria donors and non heart beating donors are particularly susceptible to injury during hypothermic preservation and may benefit from alternative methods of preservation. A switch to normothermic preservation is suggested by some, not only to prevent additional cold storage injury, but also to maintain cellular reparative mechanisms.

[0012] Therefore, there is a need to provide new composition and the method that reduce damages caused by storage and transport of organs, in order to increase success of graft.

[0013] The present invention relates to a method, preferably in vitro or ex vivo, for preservation of a tissue or an organ previously obtained from a mammal, for transplant of said tissue or organ in a recipient compatible mammal, the method comprising:

[0014] - contacting, preferably in vitro or ex vivo, said tissue or organ with a treating composition, the treating composition comprising a neddylation pathway inhibitor, for 1 to 12 hours, in order to obtain a treated tissue or organ,

[0015] said composition comprising from 1 to 100 µM of said neddylation pathway inhibitor;

[0016] - washing the treated tissue or organ with a conservation composition for removing the treating composition from the treated tissue or organ, in order to obtain a washed tissue or organ; and

[0017] - contacting washed tissue or organ with a preservation composition, in order to obtain a ready to be transplanted tissue or organ.

[0018] The inventors made the unexpected observation that inhibition of NEDD pathway surprisingly has powerful anti-inflammatory, anti-thrombotic and cytoprotective effects on endothelial cells, while maintaining good cell viability, after just a few hours of pre-treatment at 4°C, which is perfectly compatible with its use in the cold preservation of organs already implemented in the clinic.

[0019] According to the invention, organ or tissues extracted from the donor are contacted with a preservation composition of fluid containing a neddylation inhibitor, for a short time, and then the inhibitor is removed by wash, such that the organ or tissue does not contain anymore the inhibitor at the time of the graft. This transient treatment is sufficient to increase significantly cytoprotective effects of the organ or tissue that will be transplanted.

[0020] In the invention, the inhibitor, i.e. the neddylation inhibitor is contacted to the organ or the tissue during 1 to 12 hours, which means that the inhibitor is in contact with the organ or tissue during 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 hours.

[0021] In the invention, the organ or tissue can be any organ or tissue which can be transplanted, e.g., a liver, a kidney, a heart, a pancreas, a lung, small intestine, and / or skin, and the donor can be a mammal of a species different from that of the mammal recipient, or the mammal donor and the mammal recipient can be of the same species. The donor and the recipient can both be non-human mammals or humans. Alternatively, the donor can be a non-human mammal such as pig, and the recipient can be a human.

[0022] The organ or tissue transplantation includes the steps of providing an organ, tissue or cells of a donor, transplanting the organ, tissue or cells into a recipient, and before, and / or during, and / or after the step of transplanting the organ, tissue or cells into the recipient, administering to the recipient an amount of a pharmaceutical composition containing neddylation inhibitor sufficient to enhance survival and / or function of the transplanted organ, tissue or cells in the recipient.

[0023] Neddylation is a post-translational modification that transfers NEDD8 (Neural Precursor Cell-Expressed Developmentally Downregulated protein 8, NEDD8) to substrates by a three-step enzymatic that cascade like ubiquitination modification. The best-characterized substrates of neddylation are the cullin subunits of Cullin-RING ligases (CRLs), which, as the largest family of E3 ubiquitin ligases, control many important biological processes, including tumorigenesis, through promoting ubiquitination and subsequent degradation of a variety of key regulatory proteins. Recently, increasing pieces of experimental evidence strongly indicate that the process of protein neddylation modification is elevated in multiple human cancers, providing sound rationale for its targeting as an attractive anticancer therapeutic strategy.

[0024] To have an efficient effect, the neddylation inhibitor is used at a concentration of 1 to 100 µM in the treating composition. This means that the inhibitor can be used at a concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81, 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 or 100 µM in the treating composition.

[0025] The advantageous neddylation inhibitors can be chosen from: HA-1141, HA-9104, arctigenin, NAcM-OPT, NAcM-COV, DI-404, DI-1548, DI-1859, WS-383, DC-2, DN-2, SK-464, and, MLN4924.

[0026] After the incubation with the treating composition comprising the neddylation inhibitor, the organ or tissue is then washed with the composition that will be used to preserve the organ or tissue until the transplantation, i.e. is washed with the preservation composition that does not contain neddylation inhibitor.

[0027] This washing step can be repeated once or twice to be sure that no inhibitor remains in contact with the organ or tissue.

[0028] The organ or tissue is therefore placed into a fresh preservation composition for transport and storage of the organ before the transplantation.

[0029] Advantageously, the invention relates the method as described above, wherein the neddylation pathway inhibitor is an inhibitor of the NEDD8-activating enzyme.

[0030] Advantageously, the invention relates to the method as described above, wherein the neddylation pathway inhibitor is MLN4924, of formula I: (I).

[0031] Compound of formula I is MLN4924 (also known as pevonedistat - CAS 951950-33-7), a small molecule inhibitor of E1 NEDD8-activating enzyme currently in phase I / II clinical trials, exerts significant anticancer effects by inducing cell cycle arrest, apoptosis, senescence and autophagy in a cell-type and context dependent manner.

[0032] MLN4924 is a potent and selective, first-in-class inhibitor of NEDD8-activating enzyme (NAE).

[0033] NEDD8 is an ubiquitin-like protein whose activity is required to activate cullin-RING ubiquitin E3 ligases (CRLs) responsible for the ubiquitination and proteasome-dependent turnover of certain substrates with roles relevant to cellular processes important for cancer cell survival. Neddylation of CRLs is initiated by NEDD8-activating enzyme (NAE), which has become a novel target in cancer therapeutic research. MLN4924 is an analog of adenosine 5’-monophosphate that selectively inhibits NAE (IC50 = 4.7 nM). At much higher concentrations, MLN4924 inhibits the related enzymes ubiquitin-activating enzyme (UAE) and SUMO-activating enzyme (SAE) with IC50 values of 1.5 and 8.2 μM, respectively. At 0.3 μM, MLN

[0034] 4924 has been shown to disrupt CRL-mediated protein turnover leading to apoptosis in HCT-116 cells by deregulating S-phase DNA synthesis.2 Remarkably, functioning in diverse regulatory activities, proteins conjugated to UBLs like Nedd8 typically are not targeted for proteasomal degradation.

[0035] NAE inhibitor MLN4924 has potential antineoplastic activity, and is a first-in-class cancer therapy drug. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions.

[0036] More advantageously, the invention relates to the method as described above, wherein the preservation compositions are IGL-1, CELSIOR or UW SPS-1 solutions for a contact in static cold storage, and UW MPS or Perfadex for a contact in hypothermic machine perfusion.

[0037] Composition IGL-1 (i.e. modified Belzer's solution) has been modified by reversing the concentration gradient of Sodium and Potassium to make an extracellular solution (rich in Na+ and low in K+: Na-UW). HES was replaced by PEG with a molecular weight of 35,000 Daltons and used at a concentration of 0.03 mM (PEG 35).

[0038] The composition is the following one:

[0039] IGL-1PEG-35 (mM)0.03Lactobionicacid (mM)100Raffinose (mM)30MgSO4(mM)5KH2PO4(mM)25Glutathion(mM)3Adenosine(mM)5Allopurinol (mM)1Na+(mM)125K+(mM)30Osmolarity (mOsm / kg)320pH7.2-7.4

[0040] Composition CELSIOR is a Solution for the preservation of thoracic organs (heart and lungs) and abdominal organs (kidney, liver, pancreas) during a transplant procedure: from the removal of the organ from the donor, during storage and transport of the organ, to its transplantation into the recipient.

[0041] The composition is the following one:

[0042] For 1L CELSIORgmmolGlutamic acid2.94320Lactobionicacid28.66480Mannitol10.93060NaOH4100CaCl20.0370.25Glutathion0.9213KCl1.11815MgCl22.64213Histidine4.65030Osmolarity (mOsm / kg)320pH7.3

[0043] Composition UW SPS-1 is a composition intended for the flushing and cold storage of kidney, liver and pancreas organs at the time of removal from the donor in preparation for storage, transportation and eventual transplantation into a recipient

[0044] The composition is the following one:

[0045] For 1L UW SPS1gHEPES2.38Hydroxyethyl Starch50Mannitol5.4NaOH0.70CaCl20.068Glutathion0.92KH2PO43.4Beta D Glucose1.8Sodium gluconate17,45Magnesium gluconateD(-) gluconic acidhemimagnesiumsalt1.13D Ribose0.75Adenine0.68Osmolarity (mOsm / kg)310

[0046] Composition UW MPS or Belzer composition is a sterile Machine Perfusion Solution which is an isotonic non-pyrogenic solution.

[0047] The composition is the following one:

[0048] For 1L BelzerGmmolAdenine (free base)0.68 g5Calcium Chloride (dihydrate)0.068 g0.5Dextrose (+)1.80 g10Glutathione (reduced)0.92 g3HEPES (free acid)2.38 g10Hydroxyethyl Starch50.0 gN / AMagnesium Gluconate1.13 g5Mannitol5.4 g30Potassium Phosphate (monobasic)3.4 g25Ribose, D(-)0.75 g5Sodium Gluconate17.45 g80Sodium Hydroxide0.70 gN / AOsmolarity (mOsm / kg)300

[0049] Composition Perfadex

[0050] The composition is the following one:

[0051] PerfadexNa+(mM)138K+(mM)6Cl-(mM)142Mg2+(mM)0.8SO42-(mM)25Dextran(mM)50H2PO4-(mM)0.8Glucose (g)0.91Osmolarity (mOsm / kg)292pH7.4

[0052] This list is not limitative and the skilled person can easily select another composition used in the art for the purpose of collecting, storing, transporting an organ or a tissue.

[0053] More advantageously, the invention relates to the method as described above, wherein the step of contacting washed tissue or organ with a preservation composition is carried out at a temperature from 2°C to 10°C, preferably at 4°C.

[0054] It is advantageous that the contacting of the organ or the tissue be carried out at low temperature from 2°C to 10°C, which means that the temperature can be 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10°C, with a preference at 4°C.

[0055] The hypothermic environment is responsible for decreasing cellular metabolism, and the preservation solution reduces cellular metabolism and provides cytoprotection.

[0056] More advantageously, the invention relates to the method as described above, wherein the contacting, in vitro or ex vivo, of said tissue or organ with the treating composition is carried out by flushing the treating composition by hypothermic machine perfusion, or by immersing said tissue or organ in the treating composition after manual flushing.

[0057] Machine perfusion is a technique in which artificial perfusion from the blood vessels of the organ graft is continuously performed to actively control the temperature, oxygen partial pressure, chemical composition, and mechanical stress of the internal environment of the organ. This technology is already in the stage of clinical application, and clinical efficacy has been established in the field of kidney, liver, and lung transplantation. In the field of liver transplantation, machine perfusion has been used with the goal of reducing ischemia-reperfusion injury (I / R injury), preventing biliary complications, protecting the endothelium, and providing a pretransplant graft viability assessment.

[0058] These machines are well known in the art and the skilled person can easily find which machine can be used in order to carry out the process according to the invention. For instance, but not limitative, the following manufacturers propose machine perfusion devices that can be used:

[0059] AutoMate Scientific, Braile Biomedica, Bridge to Life, emka TECHNOLOGIES, Harvard Apparatus, MDE Technologies, MEDICA, OrganOx, Radnoti, TransMedics and Warner Instruments.

[0060] More advantageously, the invention relates to the method as described above, wherein the contacting, in vitro or ex vivo, of said tissue or organ with the treating composition is carried out at a temperature from 4°C to 37°C.

[0061] Whereas it is advantageous that storage and transport in the preservation composition be carried out at low temperature, the contact with the treating composition comprising the neddylation inhibitor as defined above can be carried out from 4°C to 37°C.

[0062] This means that the contact can be carried out at 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30,

[0063] 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5 and 37°C.

[0064] The invention also relates to the use of a compound of formula I: (I)

[0065] for in vitro preservation of a tissue or an organ previously obtained from a mammal.

[0066] This means that the invention relates to a compound of formula I for its use for in vitro preservation of a tissue or an organ previously obtained from a mammal.

[0067] In other words, it is disclosed to a method for preservation of an organ or a tissue comprising a step of obtaining a tissue or an organ form a mammal, and contacting said organ or tissue with a composition called treating composition containing an inhibitor of neddylation.

[0068] More advantageously, the invention relates to the use as defined above, wherein said compound is used at a concentration from 1 to 100 µM.

[0069] This means that the inhibitor can be used at a concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81, 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 or 100 µM in the treating composition.

[0070] More advantageously, the invention relates to the compound as defined above for its use as defined above, wherein said compound is used at a concentration from 1 to 100 µM.

[0071] In other words, it is disclosed to a method for preservation of an organ or a tissue comprising a step of obtaining a tissue or an organ form a mammal, and contacting said organ or tissue with a composition called treating composition containing an inhibitor of neddylation, wherein the inhibitor is present at a concentration of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81, 81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 or 100 µM in the treating composition.Brief description of the drawings

[0072] The invention will be better understood in view of the drawings and following examples:

[0073] Effects of MLN4924 on gene expression in endothelial cells. The scatter plot shows genes whose expression is deregulated in TNFα / IFNγ-stimulated HDMECs after MLN4924 pre-treatment compared to cells stimulated but not pre-treated (NT). Genes whose expression is increased (red) or decreased (blue) more than 5-fold in pretreated vs. untreated cells are indicated.

[0074] Effects of MLN4924 pre-treatment on induction of pro-inflammatory and pro-thrombotic gene expression. The expression of genes encoding adhesion molecules (ICAM1 (A), VCAM1 (B), E- SELECTINE (C)), chemokines (CCL2 (D), CCL5 (E), CXC3CL1 (F)) and pro-inflammatory cytokines (IL1B (G), IL6 (H)) and of molecules (Tissue Factor (I), SERPINE1 (J)) was analyzed by qPCR in HDMEC pretreated or not with MLN4924 (1.) 10 µM for 3h at 4°C, then stimulated with TNFα / IFNγ (2.) for 4h at 37°C (n=6).*P<0.05, **P<0.01.The histogram Y-axis represent fold change mRNA expression.

[0075] Effects of MLN4924 pre-treatment on the expression of activating, pro-thrombotic and cytoprotective molecules on the surface of endothelial cells. Expression (fold change) of the adhesion molecules ICAM1 (A) and VCAM1 (B), HLA class I (C) and class II (D), Tissue Factor (E) and the membrane complement inhibitors CD46, CD55 (F) and CD59 was analysed by flow cytometry on HDMECs pretreated (1.) or not with 10 µM MLN4924 (1.) for 3 hours at 4°C, then stimulated with TNFα / IFNγ (2.) for 24 hours at 37°C. Cell viability (G -percent of viable cells) was assessed using the fluorescent viability marker eF506 (n=7)*P<0.05, **P<0.01, ***P<0.001

[0076] Effects of MLN4924 pre-treatment on IL-6 andRANTESsecretion. Expression (concentration in pg / mL) of the pro-inflammatory cytokines and chemokines IL-6 (A) and RANTES (B) was measured by ELISA in the culture supernatant of HDMEC pretreated (1.) with MLN4924 for 3h at 4°C, then stimulated with TNFα / IFNγ (2.) for 24h at 37°C (n=6) **P<0.01.

[0077] Pre-treatment of endothelial cells with MLN4924 protects them from attack by CD8 T lymphocytes.HDMECs were pre-treated for 3h at 4°C with 10 µM MLN4924 (1.), and after washing, co-incubated at 37°C with CD8 T lymphocytes (2.) from healthy volunteers previously activated for 3 days with anti-CD3 / anti-CD28. After 24 hours of coculture, CD8 T cell activation was analysed by flow cytometry (n=8)***P<0.001. (A): ICAM1; (B): VCAM1; (C): HLA I and (D): HLA II.

[0078] Pretreatment of endothelial cells with MLN4924 inhibits their ability to activate allogeneic T cells. HDMEC were treated for 3h at 4°C with 10 µM MLN4924 (1.), then activated with TNFα / IFNγ (2.) for 48h at 37°C. After washing, these HDMECs were co-incubated with freshly isolated T lymphocytes T CD4 (A) and T CD8 (B) from healthy volunteers for a further 18h. T cell activation was assessed by analysing CD69 expression on their surface using flow cytometry (n=3). *P<0.05.

[0079] Effect of MLN4924 pre-treatment on the expression of the cytoprotective molecule HO-1. HDMECs were pretreated with 10 µM MLN4924 (1.) for 3h at 4°C and then activated with TNFα / IFNγ (2.) at 37°C. HMOX1 transcript expression (fold change) was assessed by qPCR after 4h of TNFα / IFNγ exposure (n=6). **P<0.01

[0080] Effect of MLN4924 pre-treatment on the expression of the cytoprotective molecule HO-1.HDMECs were pretreated with 10 µM MLN4924 (1.) for 3h at 4°C and then activated with TNFα / IFNγ (2.) at 37°C. HO-1 protein expression (H) was analysed by Western blot after 24h of TNFα / IFNγ exposure. β-Actin (β) was used a loading charge control. Images are representative of n=3 independent experiments.

[0081] Pretreatment of endothelial cells with MLN4924 protects them from apoptosis induced by hypothermia and hypoxia.HDMECs were incubated in UW preservation medium for 24 hours at 4°C under hypoxia (0% O2) in the presence (B) or absence (A) of 10 µM MLN4924 (hypothermic static preservation model). At the end of this incubation, the cells were washed and transferred to 37°C in complete culture medium (reperfusion model). 4 hours later, apoptosis of these cells was analysed by annexin V (1.) and 7-AAD (2.) labelling in flow cytometry (n=6). The histogram shows the percentage of cell death (Annexin V positive) for cells treated with or without of 10 µM MLN4924 (1.). **P<0.01.

[0082] represents the expression levels of 92 genes related to endothelial cell biology were

[0083] analyzed by specific qPCR arrays after 4 hours of stimulation. The scatter plot shows the relative gene expression levels in cells preconditioned with MLN4924 vs. non-preconditioned cells (NT), after TNFα / IFNγ stimulation. Genes upregulated or downregulated more than five-fold in MLN4924-preconditioned cells are shown in red and blue respectively. The table displays the fold change in mRNA expression for each of these genes in preconditioned vs. untreated HDMEC.

[0084] represents expression of genes coding for adhesion molecules (ICAM1, VCAM1, E-SELECTIN) and pro-inflammatory cytokines and chemokines (IL6, CCL5, CXC3CL1 and IL1B) was analyzed after 4 hours of stimulation by RT-qPCR. Scatter plots show the fold change in mRNA expression in 5 independent experiments (with mean ± SEM).

[0085] represents expression of adhesion molecules (ICAM1 and VCAM1) and of HLA class I and II molecules were analyzed by flow cytometry after 24 hours of stimulation. Representative histograms are shown and scatter plots show the fold change in mean fluorescence intensity (MFI) from 7 independent experiments (with mean ± SEM).

[0086] represents endothelial cell preconditioning with MLN4924 inhibits their activation for

[0087] at least 48 hours. HDMEC were pretreated or not with MLN4924 10 μM for 3 hours at 4°C and further stimulated with TNFα (100 U / mL) and IFNγ (1000 U / mL). After 48 hours, HDMEC were hit again with TNFα and IFNγ to ensure continuous stimulation. Cell surface expression of ICAM1, VCAM1, HLA I and HLA II was analyzed every 24 hours by flow cytometry, for up to 96 hours. The bar graphs represent the fold change in mean fluorescence intensity (MFI) from 3 independent experiments (with mean ± SEM).

[0088] represents release of IL6, RANTES and Fractalkine in the culture medium were quantified by ELISA after 24 hours of stimulation. Scatter plots show concentrations in pg / mL

[0089] with mean ± SEM from 6 independent experiments.

[0090] represents cell viability was assessed by flow cytometry using a fluorescent viability dye at 24 hours of stimulation. The scatter plot shows the percentage of viable cells with mean ± SEM from 7 independent experiments. *P<0.05, **P<0.01, ***P<0.001.

[0091] Preconditioning with MLN4924 inhibits human renal glomerular endothelial cell activation. Primary cultures of Human Renal Glomerular Endothelial Cells (HRGEC) were pretreated of not with MLN4924 10 μM for 3 hours ar 4°C and subsequently stimulated with

[0092] TNFα and IFNγ for 4 to 24 hours at 37°C. (A) Expression of genes coding for adhesion molecules (ICAM1, VCAM1, E-SELECTIN) and pro-inflammatory cytokines and chemokines (IL6, CCL5, CX3CL1 and IL1B) was analyzed after 4 hours of stimulation by RT-qPCR. Scatter plots show the fold change in mRNA expression in 5 independent experiments (with mean ± SEM). (B) Expression of adhesion molecules (ICAM1 and VCAM1) and of HL class I and II molecules were analyzed by flow cytometry after 24 hours of stimulation. Representative histograms are shown and the scatter plots show the fold change in mean fluorescence intensity (MFI) from 6 independent experiments (with mean ± SEM). (C) Release of IL6, RANTES and Fractalkine in the culture medium were quantified by ELISA after 24 hours of stimulation. Scatter plots show concentrations in pg / mL with mean ± SEM from 5 independent

[0093] experiments. *P<0.05, ****P<0.0001.

[0094] HDMEC pretreated for 3 hours at 4°C with 10 μM MLN4924 were co-incubated at 37°C with activated T CD4 and T CD8 lymphocytes from healthy volunteers. After 24 hours of coculture, expression of ICAM1, VCAM1, HLA I and HLA II was analyzed at the surface of HDMEC by flow cytometry. Scatter plots show the fold change in MFI from 8 independent experiments (with mean ± SEM).

[0095] HDMEC pretreated for 3 hours at 4°C with 10 μM MLN4924 and further stimulated with TNFα and IFNγ for 24 hours were co-incubated for 18 hours with freshly isolated allogenic T CD4 and T CD8 cells from healthy volunteers. T cell activation was then evaluated by analyzing CD69 expression at their surface by flow cytometry. Representative flow cytometry plots are shown, and scatter plots indicate the percentage of CD69+ T CD4 or T CD8 cells from 6 independent experiments (with mean ± SEM).

[0096] HDMEC were pretreated for 3 hours at 4°C with 10 μM or 100 μM MLN4924, stimulated with TNFα and IFNγ for 24 hours at 37°C, and further co-incubated for 1 hour at 37°C with allogenic PBMCs from healthy volunteers. After extensive washes, adherent T lymphocytes were quantified by flow cytometry. Representative flow cytometry plots are shown, and the scatter plot indicates the adhesion index of CD45+ CD3+ T cells to HDMEC from 8 independent experiments (with mean ± SEM). *P<0.05, **P<0.01.

[0097] represents endothelial cell preconditioning with MLN4924 inhibits their pro-thrombotic responses. HDMEC were pretreated with 10 μM MLN4924 for 3 hours at 4°C and further stimulated with TNFα and IFNγ at 37°C.

[0098] The mRNA expression of F3 and SERPINE-1 were analyzed by RT-qPCR after 4 hours of stimulation. Scatter plots show the fold change in mRNA expression from 6 independent experiments (with mean ± SEM).

[0099] represents expression of membrane bound tissue factor was analyzed after 24 hours of stimulation by flow cytometry. A representative histogram is shown, and the scatter plots shows the fold change in MFI from 6 independent experiments (with mean ± SEM).

[0100] After 24 hours of stimulation, HDMEC were washed and co-incubated with freshly isolated and calcein-AM stained platelets from healthy volunteers. After 1-hour, non-adherent platelets were washed away and remaining adherent platelets were visualized by immunofluorescence microscopy. Representative photomicrographs show adhering platelets (green) on HDMEC (magnification x40). The scatter plot represents the fold change in the number of adherent platelets from 6 independent experiments (with mean ± SEM). *P<0.05, ***P<0.001.

[0101] represents endothelial cell preconditioning with MLN4924 induces cytoprotective responses. HDMEC were pretreated for 3 hours at 4°C with 10 μM MLN4924 and further stimulated for 4-24 hours with TNFα and IFNγ. The expression of Heme Oxygenase was analyzed at the mRNA level by RT-qPCR after 4 hours of stimulation. The scatter plot represents the fold change in mRNA expression (with mean ± SEM) from 6 independent experiments.

[0102] Heme oxygenase expression was analyzed at the protein level by Western blot after 24 hours of stimulation. A representative blot from 3 independent experiments is shown, and the scatter plot represents the quantification of protein bands by densitometry, expressed as the ratio of HO-1 / β-actin (with mean ± SEM).

[0103] Expression of the complement inhibitory molecule CD55 was analyzed at the mRNA level by RT-qPCR after 4 hours of stimulation. The scatter plot shows the fold change in mRNA expression (with mean ± SEM) from 6 independent experiments.

[0104] Expression of CD55 was also analyzed at the protein level by flow cytometry after 24 hours of activation. A representative histogram is shown, and the scatter plot represents the fold change in MFI from 7 independent experiments (with mean ± SEM).

[0105] HDMEC were stored for 24 hours at 4°C in complete hypoxia (0% O2) in UW preservation medium containing or not 10 μM MLN4924. After a PBS wash, they were switched back to 37°C in normoxia and in complete culture medium. Cell apoptosis was evaluated after 1 hour by flow cytometry using annexin V and 7-AAD staining. Representative flow cytometry plots are shown, and the scatter plot represents the percentage of Annexin V+ apoptotic cells in 6 independent experiments (with mean ± SEM). *P<0.05, **P<0.01.

[0106] represents effects of preconditioning with NAcM-OPT and HA-9104 on the induction of proinflammatory, prothrombotic, or cytoprotective gene expression in endothelial cells, compared with preconditioning with MLN4924. The expression of genes encoding adhesion molecules (ICAM1, VCAM1, SELE (E-selectin)), proinflammatory chemokines and cytokines (CCL2, CCL5, CX3CL1, IL6), prothrombotic molecules (Tissue factor) and cytoprotective molecules (HMOX1, CD55) was analyzed by qPCR in HDMECs pretreated or not with MLN4924, NAcM-OPT, or HA-9104 at 10-100 µM for 3 hours at 4°C, then stimulated with TNFα / IFNγ for 4 hours at 37°C (n=4).

[0107] Example1:Use of MLN4924 in the protection of vascular endothelial cells against inflammation and in thepreconditioning of organs for transplantationIntroduction

[0108] Renal transplantation is the treatment of choice for end-stage renal disease, both from the patient's point of view (better quality of life, better chances of survival) and from an economic point of view (lower costs compared with long-term dialysis). However, the number of people waiting for a transplant continues to rise due to the lack of available grafts, the increasing prevalence of end-stage renal disease and the limited survival of kidney transplants. To meet this ever-increasing demand for organs, transplants from donors who have died after cardiocirculatory arrest and from donors with broader criteria (over 60s, history of diabetes or high blood pressure) are now being used. However, these "lower quality" grafts are highly sensitive to ischemia-reperfusion and are more immunogenic, and therefore have a lower survival rate. Pancreatic transplantation restores insulin secretion, and is mainly indicated in association with renal transplantation in patients with type 1 diabetes associated with stage 4 or 5 chronic renal dysfunction. Patients who have already received a first kidney transplant or who present a contraindication to pancreatic transplantation may benefit from pancreatic islet transplantation, which has been authorised for reimbursement in France since 2020 and is increasing rapidly. The pancreas is extremely sensitive to oedema and ischemia-reperfusion lesions, which have harmful effects on the graft's microvasculature and promote organ dysfunction. Early loss of pancreatic allografts is mainly due to thrombosis, which occurs in 10% of recipients and is favoured by prolonged cold ischemia times. In the case of pancreatic islet transplantation, it has been shown that short ischamia times enable them to be isolated more effectively. Improved preservation methods and preconditioning of organs prior to implantation are therefore major clinical challenges for improving the survival of kidney and pancreas transplants.

[0109] Current graft preservation techniques all involve a period of ischemia, followed by reperfusion during transplantation. Ischemia-reperfusion (I / R) lesions are aspecific lesions caused by hypoxia and the subsequent massive influx of oxygenated blood. The release of Damage Associated Molecular Patterns (DAMPs) and Hypoxia Induced Factors (HIFs) by endothelial cells (ECs) and ischemic tubular cells, as well as the expression of adhesion molecules by ECs, are associated with sterile inflammation and promote the recruitment of mediators of the adaptive immune response. The graft endothelium is at the interface between the graft and the recipient immune system, and is therefore the direct target of these immune attacks. Consequently, prolonged ischemia times and the severity of I / R lesions are associated with allograft rejection. But ECs are not only victims of I / R and the alloimmune response, they also play an active role in triggering and directing this immune response. Following stimulation by surrounding pro-inflammatory cytokines, graft ECs up-regulate HLA class I and begin to express HLA class II on their surface, secrete pro-inflammatory cytokines and chemokines, and over-express adhesion molecules. They may also present antigens and express costimulatory molecules and cytokines which help to orientate the T cell response towards a pro-inflammatory Th17 profile or a regulatory profile. Collectively, these EC responses, triggered by ischemia-reperfusion in particular, participate in 1 / infiltration of the graft by immune cells without allogeneic specificity, in particular macrophages, which in this inflammatory context will cause pathological repair with graft fibrosis, 2 / infiltration of the graft by donor-specific T lymphocytes, which promote cellular rejection, and 3 / expression by endothelial cells of antigenic targets which may promote antibody-mediated rejection.

[0110] In recent decades, graft preservation techniques have improved, with the introduction of machine perfusion of organs (as opposed to static hypothermic preservation in a preservation solution). Pre-transplant hypothermic perfusion of kidney grafts from deceased donors has beneficial effects on delayed recovery of function and long-term graft survival. Ex vivo normothermic perfusion using oxygenated perfusion solutions has the advantage of restoring a physiological environment and thus offers the possibility of better organ preservation (no ischemia), "reconditioning" (ATP replenishment) and ex vivo evaluation prior to transplantation. However, organ preservation over excessively long periods under normothermic perfusion poses problems for the elimination of toxic products derived from cellular activity. Furthermore, this procedure is costly, requires a great deal of human resources, and is still difficult to implement on a routine basis. As regards the preservation of pancreatic grafts, these techniques have had to be adapted to the fragile vasculature and low blood flow of these organs. The inventors’ team has developed pre-clinical models of pancreatic transplantation and hypothermic and normothermic perfusion of pancreatic grafts. These advances in preservation techniques pave the way for the treatment of organs prior to implantation: 'organ preconditioning'. The ex vivo injection of protective molecules into the graft has the advantage of targeting the graft alone, and in particular its endothelium, thereby overcoming many of the limitations associated with systemic drug administration (toxicity, difficulties in reaching the organ). The concept of preconditioning grafts thus opens up prospects for

[0111] 1) prevent, or at least reduce, I / R lesions, 2) reduce the immunogenicity of graft endothelium and 3) promote the repair and regeneration of organs of inferior quality.

[0112] In this work, the inventors sought to identify protective molecules capable of reducing immunogenicity and endothelial cell damage in vitro, with a view to implementing them as a preconditioning treatment for kidney and pancreas grafts prior to transplantation and reducing I / R damage and graft allogenicity. The inventors’ preliminary work has enabled us to identify a pharmacological inhibitor of neddylation, MLN4924, as a molecule of interest in this context. Neddylation is a post-translational modification of proteins during which the polypeptide NEDD8 (Neural Precursor Cell Expressed Developmentally Downregulated 8) binds to target proteins by a mechanism very similar to that of ubiquitination. This pathway has been particularly studied in tumour processes following the observation of its upregulation in various tumour cells. In 2009, it was shown that inhibition of neddylation on MLN4924 (or Pevonedistat) could suppress tumour growth, leading to several clinical trials. Some studies have also shown the importance of neddylation in regulating immune responses, in particular through its role in activating the NF-kB pathway. However, to date there is very little data on the vascular responses regulated by neddylation and the possible therapeutic value of targeting this pathway in transplantation or in inflammatory vascular pathologies in general.MethodsCell culture and study model

[0113] Primary cultures of human dermal microvascular endothelial cells (HDMEC) were obtained from Promocell and cultured in EGM-2MV complete medium (Promocell). Cells from 3 different donors were used in the inventors’ work. They were all used between passages 3 and 6.

[0114] In order to study the effects of MLN4924 pre-treatment under conditions close to those of an organ preservation system between removal and transplantation, HDMECs were incubated for 3h at 4°C in the presence or absence of 10 µM MLN4924 (Calbiochem / Sigma Aldrich) in the culture medium. The cells were then washed with PBS and cultured in complete culture medium at 37°C, in the presence or absence of 100 U / mL TNFα (recombinant human TNFα, R&D Systems) and 1000 U / mL IFNγ (recombinant human IFNγ, R&D Systems) (mimicking the inflammatory phenomena of reperfusion), for a further 4 h to 24 h depending on the analyses.Study of gene expression

[0115] To identify genes whose expression is regulated by MLN4924 pre-treatment, HDME mRNAs were isolated using the RNeasy isolation kit (Qiagen), and cDNAs were generated using M-MLV Reverse Transcriptase (Life Technologies) according to the manufacturer's instructions. qPCR plates designed to analyse the expression of 92 genes of interest related to endothelial cell biology (RT² Profiler PCR Arrays "Human endothelial cell biology", Qiagen) were then used. The results obtained were validated by quantitative RT-PCR as described below.Real-time quantitative PCR (RT-PCR)

[0116] For gene expression analyses, mRNA was extracted from HDMECs using the RNeasy isolation kit (Qiagen) and cDNAs were generated using M-MLV Reverse Transcriptase (Life Technologies) according to the manufacturer's instructions. Quantitative real-time PCR was then performed using TaqManTM gene expression assays specific for each gene of interest (Table 1) on a Viia7 thermal cycler (Applied Biosystems). The relative expression of each mRNA was normalised by the expression of HPRT in each sample and calculated using the 2-ΔΔCtmethod, as described previously.

[0117] GeneTaqManTMgene expression assay IDCCL2Hs00234140_m1IL6Hs00174131_m1CXCL8Hs00174103_m1HMOX1Hs0110250_m1FTH1Hs01694011_s1CD46Hs00611257_m1CD55Hs00892618_m1CD59Hs04991306_s1ICAM1Hs00164932_m1VCAM1Hs01003372_m1F3Hs01076029_m1CX3CL1Hs00171086_m1CCL5Hs00982282_m1SELEHs00174057_m1SERPINE1Hs00167155_m1IL1BHs01555410_m1IL7Hs00174202_m1

[0118] Table I: TaqMan probes (TaqMan Gene Expression Assays, ThermoFisher Scientific) used for RT-PCR.Flow cytometry

[0119] To analyse the expression of surface proteins on HDMECs, the cells were collected using Trypsin / EDTA and, after washing in PBS, incubated with the cell viability marker eFluorTM 506 (eBioscience) diluted 1:1000 in PBS, for 20 min at 4°C. The cells were then washed twice in FACS buffer (PBS / SVF 2% / EDTA 2mM) and incubated for 30 min at 4°C with the appropriate antibodies (Table 2) diluted 1:100 in FACS buffer. After 3 washes with FACS buffer, the cells were analysed on a FACSCanto II (BD Biosciences) using FlowJo® software (Tree Star Inc.).

[0120] AntibodiesCloneReference (Biolegend)CD46-FITCMEM-258315304CD55-APCJS11311312CD59-PEQA19A13376004HLA-A,B,C-FITCW6 / 32311404HLA-DR,DP,DQ-APCTü39361714CD54-FITC (ICAM1)HA58353108CD106-APC (VCAM1)STA305810CD142-PE (TF)NY2365204CD69-PerCPCy5.5FN50560738

[0121] Table 2: Antibodies used for flow cytometry labelling.ELISA assays (Enzyme-linked immunosorbent assays)

[0122] The secretion of IL-6, IL-8, CCL2 (MCP-1) and CCL5 (RANTES) in culture media was analysed using the specific Human DuoSet ELISA kits (R&D Systems) following the manufacturer's recommendations.Western blot

[0123] After treatment, HDMECs were lysed in RIPA buffer (Cell Signaling Technology) containing a cocktail of protease inhibitors (Thermofisher Scientific). Proteins were separated on an SDS-PAGE gel and transferred to nitrocellulose membrane. After 1h saturation in PBS / 0.5% Tween-20 / 5% BSA, the membranes were incubated overnight at 4°C with the primary antibody (Table III) diluted 1:1000 in saturation buffer. The membranes were then washed 3 times for 5 minutes in PBS / 0.05% Tween-20, then incubated for 1 hour at room temperature with the appropriate secondary antibody (Table 3) diluted 1:2000 in saturation buffer. After 3 further 5-minute washes in PBS / 0.05% Tween-20, the proteins of interest were finally revealed using PierceTM ECL Western Blotting Substrate (Thermofisher Scientific) on a ChemiDocTM MP Imaging System (Bio-Rad).

[0124] Primary antibodyCloneReferenceAntibodiessecondaryCloneReferenceHO-1 Rabbit mAbE8B7ACell Signaling Technology#26416Anti-Rabbit IgG-HRPPolyclonalCell Signaling Technology#7074β-actin mouse mAb8H10D10Cell Signaling Technology#3700Anti-mouse IgG (Fcγ) F(ab')2-POPolyclonalJackson ImmunoResearch#115-036-071

[0125] Table 3: Antibodies used for Western blot analysis

[0126] Tests for activation of endothelial cells by activated CD8 T lymphocytes

[0127] To analyse the effect of MLN4924 on endothelial cell attack by CD8 T lymphocytes, HDMECs were treated for 3 h at 4°C with 10 µM MLN4924, then washed with PBS and co-incubated with CD8 T lymphocytes from healthy EFS volunteers purified by magnetic sorting and previously activated for 3 days with anti-CD3 and anti-CD28 (1:1 ratio). After 24h of co-incubation, HDMECs were collected with Trypsin / EDTA and the expression of ICAM1, VCAM1, HLA I and HLA II on their surface was analysed by flow cytometry as previously described.

[0128] Tests for the activation of T lymphocytes by endothelial cells

[0129] To analyse the effect of MLN4924 on the ability of endothelial cells to activate allogeneic T lymphocytes (non-occupational antigen-presenting cell properties), HDMECs were treated for 3h at 4°C with 10 µM MLN4924, then washed with PBS and activated for 48h at 37°C with TNFα (100 U / mL) and IFNγ (1000 U / mL). HDMECs were then washed 2 times to remove residual TNFα and IFNγ, then co-incubated with T lymphocytes purified from the blood of healthy EFS volunteers (1:1 ratio). After 18h of co-incubation, the T lymphocytes were collected and the expression of CD69 on their surface was analysed by flow cytometry.Hypothermia- and hypoxia-induced apoptosis tests

[0130] In order to analyse the effect of MLN4924 on endothelial cell death induced by hypothermia and hypoxia, HDMEC were incubated for 24 hours at 4°C in hypoxia bags (0% O2, Anaerocult® A, Merck) in UW preservation medium (Belzer MPS® UW) and in the presence or absence of 10 µM MLN4924 (hypothermic static preservation model). After 24 hours of storage, the cells were washed with PBS and incubated at 37°C in complete culture medium (reperfusion model). After 4 h of incubation, apoptosis was analysed by flow cytometry labelling with Annexin V-APC and 7-AAD (BD Biosciences).Results

[0131] Pretreatment of endothelial cells with MLN4924 regulates the expression of inflammatory, thrombotic and cytoprotective genes

[0132] To identify genes whose expression might be regulated in endothelial cells (ECs) by MLN4924 pre-treatment during hypothermic perfusion, the inventors treated HDMECs for 2h at 4°C with 10 µM MLN4924, then the cells were washed and stimulated with TNFα / IFNγ for a further 4h at 37°C (a model of inflammation at the time of transplantation). The expression levels of 92 genes relating to endothelial cell biology were then analysed using specific qPCR arrays (). The inventors identified a panel of genes whose expression is strongly repressed by MLN4924 pre-treatment under inflammatory conditions, in particular genes encoding adhesion molecules (ICAM1, VCAM1, E-SELECTINE), pro-inflammatory cytokines and chemokines (CCL2, CCL5, CX3CL1, IL6, IL7, IL1B), and pro-thrombotic molecules (F3, SERPINE1). Conversely, some genes were identified as overexpressed following MLN4924 pre-treatment, notably the HMOX1 gene encoding Heme Oxygenase 1 (HO-1), which is well known for its cytoprotective properties against oxidative stress in particular.

[0133] These results were validated by qPCR in 6 new independent experiments (), and confirm the ability of cold MLN4924 pre-treatment to block the induction of pro-inflammatory and pro-thrombotic gene expression in response to TNFα / IFNγ identified in the qPCR arrays.

[0134] Pre-treatment of endothelial cells with MLN4924 blocks their pro-inflammatory and pro-thrombotic responses

[0135] The inventors then analysed the effect of MLN4924 pre-treatment on endothelial cell activation in response to TNFα / IFNγ at the protein level. HDMEC were pre-treated with 10 µM MLN4924 for 3h at 4°C, then after washing, stimulated with TNFα / IFNγ for 24h at 37°C, and analysed by flow cytometry (). The inventors showed that MLN4924 pre-treatment blocked the induction of the expression of the adhesion molecules ICAM1 and VCAM1 on the surface of ECs, reduced the expression of HLA class I and blocked the induction of HLA class II. Induction of membrane Tissue Factor, a pro-thrombotic molecule, following EC activation was also blocked by MLN4924 pre-treatment. The inventors also analysed the expression of the complement inhibitors CD46, CD55 and CD59 on the surface of ECs and observed an increase in CD55 expression in cells pre-treated with MLN4924, suggesting that this treatment could reduce the cytotoxicity of antibodies directed against the graft. It is important to note that the viability rate of ECs following pre-treatment with MLN4924 was not reduced compared with untreated cells, suggesting low toxicity of this treatment on the endothelium under these treatment conditions.

[0136] The inventors also measured the concentrations of IL-6 and CCL5 (RANTES) in the culture medium of ECs pretreated for 3 h cold with MLN4924 and then stimulated for 24 h at 37°C with TNFα / IFNγ, and showed that pretreatment with MLN4924 blocks the induction of secretion of these 2 pro-inflammatory molecules (), as previously suggested at transcriptional level.

[0137] Pre-treatment of endothelial cells with MLN4924 protects them from attack by CD8 T lymphocytes

[0138] The inventors wanted to find out whether pre-treatment of endothelial cells with MLN4924 could also block their activation by activated CD8 T lymphocytes. To do this, HDMECs were treated with 10 µM MLN4924 for 3 h at 4°C, then incubated with CD8 T lymphocytes from healthy volunteers previously activated for 3 days with anti-CD3 + anti-CD28. After 24 hours, the expression of ICAM1, VCAM1, HLA I and HLA II on the surface of HDMECs was analysed by flow cytometry. As shown in, the inventors demonstrated that pre-treatment of endothelial cells with MLN4924 inhibited their activation by activated CD8 T cells.

[0139] MLN4924 inhibits the ability of endothelial cells to activate allogeneic T lymphocytes

[0140] In the presence of inflammatory stimuli, activated endothelial cells overexpress HLA class I and begin to express HLA class II, which confers on them the properties of non-professional antigen-presenting cells, and therefore enables them to activate allogeneic T lymphocytes. To determine the effect of MLN4924 pre-treatment on these properties, the inventors treated HDMECs with 10 µM MLN4924 for 3h at 4°C, then after PBS wash the inventors activated these HDMECs with TNFα / IFNγ for 48h at 37°C. After washing with PBS, T lymphocytes from healthy volunteers were added to each well for 18h of coculture, and the activation of these T lymphocytes was then analysed by CD69 expression on their surface using flow cytometry. The inventors showed that pre-treatment of endothelial cells with MLN4924 significantly reduced the activation of allogeneic T lymphocytes ().

[0141] MLN4924 induces expression of the cytoprotective molecule HO-1

[0142] The inventors then sought to determine whether the HO-1 (Heme Oxygenase 1) protein, well known for its cytoprotective effects, was increased in ECs following their pre-treatment with MLN4924, as suggested by the results of qPCR arrays. HDMECs were treated with 10 µM MLN4924 for 3h at 4°C, washed and then stimulated with TNFα / IFNγ for 4h to 24h, and mRNA and HO-1 protein expression were assessed by qPCR and Western blot respectively. As shown inFigures 7 and 8, the inventors observed an increase in the expression of the HMOX1 transcript and its HO-1 protein following pre-treatment with MLN4924.

[0143] The treatment of endothelial cells with MLN4924 during their preservation in hypothermia and hypoxia has protects against apoptosis.

[0144] In order to reproduce in vitro preservation and reperfusion as closely as possible, the inventors incubated HDMEC in UW preservation medium (used for clinical graft preservation) at 4°C for 24 hours, in hermetically sealed bags containing 0% O2 (hypothermic static preservation model). At the end of these 24 hours of 'preservation', the cells were incubated in complete culture medium at 37°C and normoxia (reperfusion / transplantation model). Following this transition to 37°C normoxia, significant cell death was rapidly observed macroscopically, and confirmed by annexin V labelling of apoptosis after 4 h of "reperfusion" (). In contrast, when HDMECs were incubated at 4°C, 0% O2 in the presence of 10 µM MLN4924, a sharp decrease in their entry into apoptosis was observed following 'reperfusion' ().

[0145] Conclusion

[0146] In this work, the inventors used an in vitro microvascular endothelial cell treatment model to reproduce the conditions of cold organ preservation (3h at 4°C) followed by warm reperfusion (37°C) in the presence of inflammatory factors (transplantation model). This enabled us to test the effects of adding a neddylation inhibitor, MLN4924, during the cold preservation phase, on later endothelial responses following stimulation by the inflammatory factors TNFα and IFNγ or by activated CD8 T lymphocytes. The results obtained show that this 3h pre-treatment at 4°C with MLN4924 blocks the induction of the expression of numerous pro-inflammatory molecules, namely adhesion molecules (ICAM1, VCAM1, E-selectin), HLA class I and II molecules, the proinflammatory cytokines IL6 and IL1β, and the proinflammatory chemokines CCL2 (MCP-1), CCL5 (RANTES) and CX3CL1 (Fractalkine). One of the consequences of this is a reduction in the activation of allogeneic T lymphocytes by endothelial cells pre-treated with MLN4924. MLN4924 pre-treatment also inhibits the expression of the prothrombotic molecules Tissue Factor and Serpin 1. In addition, the inventors showed that MLN4924 induced expression of the cytoprotective molecule HO-1 and the membrane complement inhibitor CD55. These effects were not accompanied by a significant decrease in cell viability, suggesting low toxicity of MLN4924 on endothelial cells under these treatment conditions. On the contrary, pre-treatment of endothelial cells with MLN4924 even protected them from cell death induced by hypothermia and hypoxia in vitro. Overall, these results suggest that the addition of MLN4924 to the graft preservation fluid during the preservation phase under hypothermic conditions could make it possible to counteract the inflammatory and thrombotic responses of these cells in the days following transplantation and to combat ischemia-reperfusion damage, with potentially beneficial longer-term effects on the follow-up of the transplants.

[0147] Functional tests must now be carried out to verify the anti-inflammatory effect of MLN4924 pre-treatment (PBMC-endothelial cell adhesion tests), its anti-thrombotic effects (platelet-endothelial cell adhesion tests), and its ability to protect endothelial cells against complement attack (C3 deposition tests, alloantibody and complement-mediated cytotoxicity tests).

[0148] Finally, the inventors will test the therapeutic potential of MLN4924 in a preclinical porcine model of kidney and pancreas preservation. This model involves harvesting organs from pigs after a 30-minute (pancreas) or 1-hour (kidney) phase of warm ischemia by clamping the artery (donation model after cardio-circulatory arrest), then perfusing the organs for 12-24 hours on a hypothermic perfusion machine using a preservation solution containing or not 10-100 µM MLN4924. At the end of this preservation / preconditioning period, the organs can be:1) evaluated on a normothermic oxygenated perfusion machine with the donor's blood ("self-perfusion") or that of a third party ("allo-perfusion") for 6 hours, which will enable the effects of MLN4924 preconditioning on ischemia-reperfusion lesions and the immunogenicity of the graft endothelium to be evaluated at an early stage. The advantage of this method is that it reduces the number of animals used, to avoid complications related to the surgical procedure during transplantation, and to perform close biopsies during initial reperfusion;2) be re-transplanted to the donor animal (autotransplantation) in order to be able to assess the effects of MLN4924 preconditioning on ischemia-reperfusion lesions and their consequences on the graft's return to function and on its chronic loss of function in the few months following transplantation;3) be transplanted to a third animal (allotransplantation) to assess the effects of MLN4924 preconditioning on graft immunogenicity and alloimmune response in the few months following transplantation.

[0149] Ultimately, this work makes it possible to demonstrate the therapeutic role of MLN4924 for the preconditioning of kidney and pancreas grafts prior to transplantation to prevent ischemia-reperfusion lesions and graft allogenicity.

[0150] Similar results were obtained by using MLN4924 at a temperature varying from 25°C to 37°C, preferably varying from 34°C to 37°C.

[0151] Example 2:Neddylation blockade protects endothelial cells against inflammation andischemia-reperfusion injuries

[0152] To study the potential of neddylation blockade as a preconditioning strategy, The inventors used an in vitro model of microvascular endothelial cells (ECs) treated in conditions close to those of an organ preservation system. ECs were subjected to hypothermic preservation (4°C) in the presence of the neddylation inhibitor MLN4924, and following rewarming (37°C), stimulated with TNFα and IFNγ to mimic sterile inflammation at reperfusion.

[0153] By improving patient quality of life and survival, solid organ transplantation stands as the elective treatment for patients suffering from end-stage organ failure. However, despite ongoing advances in the field, the availability of grafts remains a matter of concern. To solve the current organ shortage, extended criteria donors (ECD) and donors after circulatory death (DCD) have to be used, despite a higher susceptibility of these grafts to ischemia-reperfusion injuries (IRI). Thus, improving preservation strategies of these vulnerable organs appears necessary.

[0154] Lately, significant efforts have been made to improve graft preservation strategies, not only to

[0155] maintain organ viability, but also to improve their quality. Pharmacological supplementation of the preservation solution with protective molecules could present the advantage of targeting the graft only, especially its endothelium, avoiding many limitations of systemic drug delivery (toxicity, failure to reach the graft). This so-called “graft preconditioning” provides the opportunity to prevent or at least reduce IRI and dampen the immunogenicity of the graft endothelium. Combined with the current standards of hypothermic preservation methods, graft preconditioning by “supplemented” preservation fluid could represent an effective and easy strategy for graft quality improvement.

[0156] In this work, the inventors have studied MLN4924, a neddylation inhibitor, as a potential pharmacologic agent for graft preconditioning. Neddylation is a type of protein post-translational modification in which the ubiquitin-like protein NEDD8 (Neural precursor cell Expressed, Developmentally Downregulated protein 8) binds to target proteins and directs them to proteasome for degradation, through a process analogous to ubiquitination. Neddylation has been particularly studied in cancer and MLN4924, a first-in-class small molecular inhibitor of this pathway has been previously investigated in several clinical trials. There is evidence that neddylation blockade by MLN4924 abrogates NF-kB and mTOR signaling, key pathways that notably drive endothelial cell pro-inflammatory responses. MLN4924 has also been shown to induce HIF-1α accumulation14, suggesting that it may improve the capacity of endothelial cells to control oxidative stress. Thus, considering ischemia-reperfusion injury pathophysiology, the use of MLN4924 could be considered in transplantation, for promoting cytoprotection against ischemia by impairing proinflammatory pathways.

[0157] Here, the inventors describe for the first time the use of MLN4924 in an in vitro cold ischemia-reperfusion model which will pave the way for its use for solid organ transplant preconditioning.Materials and methods

[0158] Antibodies and reagents

[0159] The antibodies and qPCR probes used in this work are listed in Supplementary Tables 4 to 6.

[0160]

[0161] Table 4: Listing of monoclonal antibodies used for flow cytometry

[0162]

[0163] Table 5: Listing of antibodies used for Western blot

[0164]

[0165] Table 6: Listing of TaqMan probes used for RT-qPCR analyzes (ThermoFisher Scientific)Cell culture and study model

[0166] Primary cultures of human microvascular dermal endothelial cells (HDMEC) from single donors were purchased from Promocell and primary cultures of human renal glomerular endothelial cells (HRGEC) from single donors were purchased from iCelltis. Both were cultured in complete endothelial growth medium EGM-2MV (Promocell). HDMEC from 3 donors and HRGEC from 2 donors were used in this work, between passages 3 and 6.

[0167] To study the effects of a pretreatment with MLN4924 in conditions close to those of a graft preservation system, HDMEC and HRGEC were incubated for 3 hours at 4°C with 10 μM to 100 μM MLN4924 (Calbiochem), or with DMSO as the vehicle control (indicated in the figures as NT for “no treatment”). Cells were then washed with PBS, switched to 37°C in complete culture medium, and stimulated or not with 100 U / mL TNFα (recombinant human TNFα, R&D Systems) and 1000 U / mL IFNγ (recombinant human IFNγ, R&D Systems) in order to mimic the inflammatory reaction that occurs following reperfusion, for 4, 24 and 48 additional hours depending on the analyses.Gene expression studies

[0168] To identify genes whose expression is regulated by the MLN4924 pretreatment, mRNA of HDMEC was extracted using the RNeasy isolation kit (Qiagen) and cDNA were generated using the M-MLV Reverse Transcriptase (Life Technologies) according to the manufacturer’s instructions. qPCR plates designed to analyze the expression of 92 genes of interest related to endothelial cell biology (RT² Profiler PCR Arrays “Human endothelial cell biology”, Qiagen) were used to examine endothelial cell responses.

[0169] Relative gene expression levels were evaluated using ACTB, B2M, GAPDH, HPRT1 and RPL10 as references, according to the 2-ΔΔCtmethod as previously described. Results obtained were validated by RT-qPCR as described below.Quantitative real-time PCR (RT-qPCR)

[0170] For gene expression analyses, mRNA of HDMEC and HRGEC was extracted using the RNeasy isolation kit (Qiagen) and cDNA generated using the M-MLV Reverse Transcriptase (Life Technologies) according to the manufacturer’s instructions. Real-time quantitative PCR were then performed using TaqManTMgene expression assays specific to each gene of interest (Table 7) on a Viia7 thermocycler (Applied Biosystems). Relative expression levels of each gene were calculated using HPRT1 as reference, according to the 2-ΔΔCtmethod.Flow cytometry

[0171] HDMEC and HRGEC were collected with Trypsin / EDTA, washed with PBS and incubated for 20 min at 4°C with the Cell Viability Dye eFluorTM506 (eBioscience) diluted at 1:1000 in PBS. After washing, cell surface molecules were stained using specific fluorescent antibodies (Table 5) for 30 min at 4°C. Cells were acquired with a BD FACSCanto II flow cytometer (BD Biosciences) and results were analyzed using the FlowJo® 10.10.0 software (BD Biosciences).Enzyme-linked immunosorbent assays

[0172] Secretion of IL-6, RANTES and Fractalkine were analyzed in culture supernatants using the specific Human DuoSet ELISA kits (R&D Systems) according to the manufacturer’s instructions.Western blot

[0173] HDMEC were lysed with RIPA Buffer (Cell Signaling Technology) containing a cocktail of protease inhibitors (ThermoFisher Scientific). Proteins were separated on a SDS polyacrylamide gel and transferred onto nitrocellulose membranes. After blocking 1 h with PBS / 0.1% Tween-20 containing 5% BSA, membranes were incubated overnight at 4°C with primary antibodies, washed, and incubated with specific secondary peroxidase-coupled antibodies (Table 6). Proteins of interest were finally revealed using the Enhanced Chemiluminescent (ECL) substrate (Pierce) on a ChemiDocTM MP imaging system (BIO-RAD). Quantification was performed using the ImageJ software, by measuring the mean grey value of each band.T lymphocyte-mediated endothelial cell aggression assays

[0174] Blood from healthy volunteers was collected in EDTA tubes at the Etablissement Français du Sang (EFS, Nantes, France) with written informed consent. PBMCs were separated from blood samples on a Ficoll gradient layer according to the manufacturer’s recommendations and untouched CD4 and CD8 T lymphocytes were isolated by magnetic separation on an AutoMACS cell separator (Miltenyi Biotec), using the CD4+ T Cell Isolation kit and the CD8+ T cell Isolation kit respectively (Miltenyi Biotec) according to the manufacturer’s protocol. These cells were activated for 3 days with human T-activator CD3 / CD28 Dynabeads (Gibco) at a 1:1 ratio in complete RPMI medium. HDMEC were pretreated with 10 μM MLN4924 for 3 h at 4°C in EGM-2MV, washed with PBS, and co-incubated at 37°C for 24 h with activated CD4 or CD8 T cells at a 1:1 ratio in EGM-2MV medium. HDMEC were then washed extensively

[0175] to remove T cells, collected by Trypsin / EDTA digestion, and their activation was assessed by analyzing the expression of ICAM-1, VCAM-1, HLA class I and HLA class II by flow cytometry as described above.Endothelial cell-mediated T lymphocyte activation assays

[0176] To assess the effect of MLN4924 on the capacity of endothelial cells to activate allogenic T lymphocytes (non-professional antigen-presenting cell properties), HDMEC were treated for 3 h at 4°C with 10 μM MLN4924, washed with PBS and activated for 24h at 37°C with TNFα (100 U / mL) and IFNγ (1000 U / mL).

[0177] HDMEC were then washed to remove residual TNFα and IFNγ, and co-incubated with T lymphocytes (1:1 ratio) freshly isolated from the blood of healthy volunteers on an AutoMACS cell separator and a Human pan T cell isolation kit (Miltenyi Biotec), as per manufacturer’s instructions. After 18 h of co-incubation, T lymphocytes were collected and the expression of the activation marker CD69 was analyzed at their surface by flow cytometry.Adhesion of T lymphocytes to endothelial cells

[0178] HDMEC pretreated with 10 μM or 100 μM of MLN4924 for 3h at 4°C, and further activated for 24 h at 37°C with TNFα (100 U / mL) and IFNγ (1000 U / mL), were co-incubated for 1h with PBMCs from healthy volunteers isolated on a Ficoll gradient layer from blood samples collected at the Etablissement Français du Sang (EFS, Nantes, France) (Ratio of 5 PBMC for 1 HDMEC). Nonadherent PBMCs were removed by 5 PBS washes, and HDMEC and adherent cells were harvested using Trypsin / EDTA and analyzed by flow cytometry using anti-CD3, anti-CD45 and anti-CD31 fluorescent antibodies (Table 5). Ten thousand cells per sample were analyzed and the adhesion index was calculated as the ratio of the percentage of CD45+ CD3+ T cells within each group, compared with the untreated group (no MLN4924 pretreatment and no TNFα / IFNγ activation).Platelet adhesion assay

[0179] Human platelets were isolated from blood samples from healthy volunteers. Blood was drawn in Vacutainer tubes containing sodium citrate, acid citrate and glucose (ACD) and centrifuged at 180 g for 5 min at room temperature (RT) without brake to separate the platelet-rich plasma. Platelet-rich plasma was diluted in Tyrode buffer and further centrifuged at 150 g for 5 min at RT without brake to eliminate potential residual red blood cells, and finally at 500 g for 5 min at RT without brake to pellet platelets. Platelets were stained with 2.5 μmol / L calcein acetoxymethyl ester (calcein-AM, Life Technologies) for 15 min at 37°C in the dark. After washing with Tyrode buffer, platelets were co-incubated with HDMEC in complete EGM-2MV culture medium for 1 hour at 37°C. After 5 washes in PBS, adherent platelets were visualized by fluorescent microscopy on an Olympus IX71 inverted microscope (x40 magnification) with an Olympus DP72 camera using CellSens Viewer acquisition software (Olympus), and quantified using ImageJ software.Hypothermia and hypoxia-induced endothelial apoptosis

[0180] HDMEC were incubated in UW preservation medium (Belzer MPS® UW) supplemented or not with 10 μM MLN4924, for 24h at 4°C in incubation bags containing a reagent mixture that chemically binds the oxygen quickly to create a 0% O2atmosphere (AnaerocultTM A mini, Millipore), as model of static cold storage. After these 24h of storage, HDMEC were washed with PBS and incubated at 37°C in complete culture medium EGM-2MV as a model of reperfusion. After 4h, cell apoptosis was analyzed by flow cytometry using Annexin V-APC and 7-AAD staining (BD Biosciences).Statistical analyses

[0181] All data are presented as mean ± SEM. Statistical analyzes were performed using the Wilcoxon test, and P values < 0.05 were considered significant.Results

[0182] Preconditioning of endothelial cells with MLN4924 inhibits their activation and proinflammatoryresponses

[0183] To identify genes whose expression might be regulated in endothelial cells by a treatment with

[0184] MLN4924 during the hypothermic preservation of an organ, the inventors set up an in vitro model in which HDMEC were treated for 3h at 4°C with 10 μM MLN4924, and further washed and stimulated with TNFα / IFNγ for 4h at 37°C. The expression level of 92 genes related to endothelial cell biology were then analyzed using targeted qPCR arrays, which led to the identification of a panel of genes whose expression was strongly repressed by MLN4924 pretreatment in inflammatory conditions, notably genes encoding adhesion molecules (ICAM1, VCAM1, E-SELECTIN) and pro-inflammatory chemokines and cytokines (CCL2, CCL5, CX3CL1, IL6, IL1B) (andTable7).

[0185]

[0186]

[0187] Table 7: Analysis of genes regulated by MLN4924 in activated endothelial cells. HDMEC were treated with 10 μM MLN4924 for 3 hours at 4°C, subsequently activated with TNFα and IFNγ for 4 hours at 4°C, and the expression levels of 92 genes related to endothelial cell biology were analyzed by specific qPCR arrays. The table summarizes the fold change in expression of each gene vs. untreated and unstimulated HDMEC. Genes upregulated or downregulated more than 5-fold between MLN4924-treated HDMEC and untreated HDMEC upon activation are shown in bold and grey respectively.

[0188] These results were validated by qPCR in 5 independent experiments ().

[0189] The effect of the MLN4924 pretreatment on endothelial cell activation was then analyzed at

[0190] the protein level. HDMEC were treated for 3h at 4°C with 10 μM MLN4924, and further activated for 24h at 37°C with TNFα / IFNγ. Flow cytometry analyzes showed that the MLN4924 pretreatment drastically inhibited the induction of ICAM1, VCAM1, HLA class I and HLA class II expression at the surface of HDMEC after 24h of stimulation (), which was still the case after 48 to 72h (). MLN4924 pretreatment also abrogated the release of IL6, CCL5 / RANTES and CX3CL1 / Fractalkine by HDMEC in the culture medium ().

[0191] Importantly, HDMEC viability was not significantly decreased following MLN4924 pretreatment as compared to untreated cells, suggesting a low toxicity of this molecule on the endothelium in these treatment conditions (). All these results except for IL6 regulation were confirmed in primary human renal glomerular endothelial cells (HRGEC) ().

[0192] Altogether, these results demonstrate that neddylation inhibition during hypothermic preservation may dampen EC inflammation during the reperfusion phase.

[0193] Preconditioning of endothelial cells with MLN4924 inhibits lymphocyte-endothelial interactions

[0194] To determine whether MLN4924 could prevent endothelial cell aggression by activated T lymphocytes, ECs were treated with 10 μM of MLN4924 for 3h at 4°C, and subsequently co-incubated at 37°C with purified CD4 T cells or CD8 T cells from healthy volunteers that had been previously activated for 3 days by anti-CD3 and anti-CD28 stimulation. After 24h, the activation of ECs was evaluated by flow cytometry. As illustrated in, pretreatment of ECs with MLN4924 inhibited the induction of ICAM1, VCAM1, HLA class I and HLA class II by both activated CD4 and CD8 T lymphocytes, suggesting that this preconditioning was able to protect them against T cell aggression.

[0195] The inventors also investigated whether MLN4924 preconditioning modulates the antigen-presenting function of ECs. ECs were treated with MLN4924, activated with TNFα / IFNγ, and after 24h of

[0196] stimulation, they were co-cultured with freshly isolated allogenic T lymphocytes for an additional 24h.

[0197] The activation of CD4+ and CD8+ T cells was then evaluated by analyzing the expression of CD69 at their surface by flow cytometry. As shown in, ECs activated with TNFα / IFNγ were able to activate allogenic T lymphocytes, as observed by the induction of CD69 on a subset of alloreactive cells, although it was not sufficient to induce T cell proliferation (data not shown), probably because of the lack of adequate costimulatory molecules on ECs. However, pretreatment of ECs with MLN4924 significantly reduced CD69 expression in both CD4 and CD8 T cells (), suggesting impaired antigen-presenting capacity of ECs following neddylation inhibition.

[0198] Given the fact that ECs pretreated with MLN4924 did not upregulate adhesion molecules upon

[0199] TNFα and IFNγ stimulation, the inventors next evaluated whether this resulted in the inhibition of T lymphocyte adhesion to ECs. To do so, HDMEC pretreated for 3h at 4°C with MLN4924 at 10 μM or 100 μM and subsequently stimulated for 24h with TNFα / IFNγ were co-incubated with freshly isolated human PBMC from healthy volunteers. After 1h of co-incubation, nonadherent PBMC were removed by extensive PBS washing, and adherent leukcytes were collected and analyzed by flow cytometry. As illustrated in, pretreatment of ECs with MLN4924 significantly inhibited T lymphocyte adhesion to these cells upon activation, and even completely abrogated it at high doses (100 μM).

[0200] Collectively, these results highlight the ability of MLN4924 preconditioning to impair T cell-endothelial interactions and protect ECs from immune activation.

[0201] Preconditioning of endothelial cells with MLN4924 inhibits their prothrombotic responses

[0202] The inventors next studied whether MLN4924 pretreatment could prevent EC prothrombotic responses, suggested by their first qPCR screening that pointed at a downregulation of Tissue Factor (F3) and Plasminogen Activator Inhibitor 1, PAI-1 (SERPINE1) gene expression in MLN4924 treated cells (). This finding was confirmed by qPCR, which showed that MLN4924 pretreatment completely blocked the induction of Tissue Factor and PAI-1 mRNA expression upon TNFα / IFNγ stimulation of ECs (). The inventors also analyzed the expression of membrane bound Tissue Factor at EC surface by flow cytometry and found that its induction by TNFα / IFNγ stimulation was completely abrogated by MLN4924 pretreatment (). To determine whether this regulation had functional antiaggregant consequences, the inventors analyzed platelet adhesion to ECs in vitro, and found that MLN4924 pretreatment resulted in a significant decrease in platelet adhesion compared with untreated cells (). Collectively, these data demonstrate that MLN4924 preconditioning effectively suppresses the prothrombotic phenotype of ECs, supporting its potential for the prevention of thrombosis after transplantation.

[0203] Preconditioning of endothelial cells with MLN4924 induces cytoprotective responses

[0204] The inventors’ first qPCR screening of genes regulated in ECs upon MLN4924 pretreatment identified HMOX1, coding for Heme Oxygenase 1 (HO-1), a well-known cytoprotective and anti-oxidant molecule, as markedly upregulated in treated cells (). The inventors confirmed this upregulation at the mRNA level by additional qPCR analyzes () and at the protein level by Western blot (). In addition, the inventors analyzed the expression of complement regulatory molecules in ECs preconditioned with MLN4924 and found that CD55 was significantly upregulated at the mRNA level () and at the protein level as shown by flow cytometry ().

[0205] Finally, the inventors studied whether the preconditioning of ECs with MLN4924 could prevent damages to these cells induced by cold storage and hypoxia. To do so, ECs were incubated for 24h at 4°C in hypoxia (0% O2) in UW preservation medium, and subsequently washed and switched back to 37°C in normoxia and in complete endothelial medium, as a model of static cold storage followed by reperfusion. EC apoptosis was then assessed using Annexin V and 7-AAD staining by flow cytometry.

[0206] The inventors found that the addition of MLN4924 at 10 μM in the preservation medium during the cold storage phase was able to markedly decrease EC apoptosis (). This suggests that the preconditioning of grafts with MLN4924 during cold storage will have implications for the prevention of endothelial damage associated with ischemia-reperfusion injuries.Discussion

[0207] Improving graft preservation remains a major clinical challenge in the current context of a persistent shortage of organs available for transplantation that has led to increase the use of organs from extended criteria donors and from donors after cardiac arrest – both highly sensitive to ischemia-reperfusion injuries. These past two decades, organ transplant preservation techniques have been improved, with the introduction of machine perfusion of organs (as opposed to static cold storage in a preservation solution). Hypothermic machine perfusion of transplants from deceased donors has beneficial effects on delayed graft function or early dysfunction and on long term graft survival. Ex vivo normothermic machine perfusion using oxygenated perfusion solutions has also been tested and has the advantage of restoring a physiological environment, and thus offers the possibility of better preserving organs (less ischemia), “reconditioning” them (ATP replenishment) and evaluating them ex vivo prior to transplantation. However, keeping organs under normothermic perfusion for long periods poses problems for the elimination of toxic products derived from cellular activity. Besides, this procedure is costly, requires a lot of human resources, and is still difficult to implement on a

[0208] routine basis. An attractive strategy to further improve graft preservation consists in the treatment of organs with protective agents during the preservation phase, so called “graft preconditioning”. The addition of protective pharmacological molecules to the preservation solution is easy to implement, and makes it possible to target only the graft, notably its endothelium, thus avoiding numerous off- target effects. Here, the inventors identify MLN4924, a small molecular inhibitor of the neddylation pathway, as a promising candidate for endothelial protection with strong potential for clinical implementation in graft preconditioning.

[0209] In this example, the inventors used an in vitro model of treatment of microvascular endothelial cells in order to reproduce the conditions of a hypothermic organ preservation (preconditioning at 4°C), followed by warm reperfusion (37°C) in the presence of the inflammatory factors TNFα and IFNγ (model of sterile inflammation following transplantation). This model enabled the inventors to evaluate the impact of the neddylation inhibitor MLN4924 as a preconditioning agent during the cold preservation phase on later endothelial responses. While the current average time of hypothermic preservation for organ transplants lasts from 2 to 14 hours depending on the organ, we first used a relatively short preconditioning time of only 3h at 4°C with 10 μM MLN4924 in our molecular analyses, which was defined in preliminary experiments as the optimal treatment dose and duration to obtain a maximal anti-inflammatory effect of MLN4924 on endothelial cells, with low associated cell death (data not shown). Indeed, longer incubation times of ECs in hypothermia resulted in increased cell death in the absence of MLN4924 treatment in vitro, compromising mRNA and protein analyses due to the poor quality of samples from untreated cells. However, it is very likely that longer MLN4924 preconditioning of organs will have equally powerful anti-inflammatory effects on graft endothelial cells.

[0210] One of the most striking effects of MLN4924 in the inventors’ model is the strong inhibition of endothelial cell activation and inflammation. Indeed, pretreatment of endothelial cells with 10 μM MLN4924 for 3 hours at 4°C prior to stimulation with TNFα and IFNγ was able to completely abrogate the induction of the adhesion molecules ICAM1, VCAM1 and E-SELECTIN, to inhibit the upregulation of HLA class I and the induction of HLA class II at the surface of ECs, and to strongly decrease the expression and release of IL6, RANTES and Fractalkine. MLN4924 preconditioning also protected ECs from T CD4 and T CD8 lymphocyte-mediated aggression. As a result, preconditioning with MLN4924 could prevent the adhesion of T lymphocytes to ECs, and markedly inhibited EC-mediated allogenic T lymphocyte activation. These anti-inflammatory effects are certainly mostly due to the blockade of the NF-kB pathway by MLN4924, which is a major signaling pathway driving endothelial cell activation.

[0211] Importantly, these effects are obtained after a simple short pretreatment of cells at 4°C and last for as long as 48 hours, even though MLN4924 was washed away after pretreatment. This suggests that graft preconditioning with MLN4924 will be able to block sterile inflammation following reperfusion at transplantation. This is particularly interesting since this short pretreatment modality shows little toxicity on ECs, in contrast to prolonged treatments with MLN4924 at 37°C that significantly impaired EC viability (data not shown).

[0212] Another key effect of MLN4924 in our studies is its ability to protect ECs from hypothermia and hypoxia induced apoptosis. Indeed, the addition of MLN4924 in the preservation medium during incubation of ECs for 24h at 4°C under hypoxic conditions (0% O2) before switching them back to 37°C in normoxic conditions was able to strongly decrease subsequent EC apoptosis. This finding supports the potential of MLN4924 preconditioning to confer protection against ischemia-reperfusion injuries. This was rather unexpected since MLN4924 has been shown to block both NF-kB26 and mTOR signaling, two pathways classically known to drive EC survival, by inhibiting the neddylation and further degradation of their intrinsic regulators DEPTOR15 and IkBα respectively. However, studies have also shown that hypoxia-induced endothelial apoptosis was due to NF-kB-mediated Bcl-2 suppression, which could explain in part the anti-apoptotic effect of MLN4924 in this setting. In addition, the inventors have found that preconditioning of ECs with MLN4924 resulted in a marked and sustained upregulation of

[0213] the protective antioxidant molecule HO-1, which is well known to protect ECs against apoptosis, and thus probably greatly participates in the mechanisms of EC protection against cell death.

[0214] Another interesting effect of MLN4924 preconditioning was the induction of the complement inhibitor CD55 (DAF) at the surface of ECs. This suggests that MLN4924 might also protect ECs against complement-mediated attacks at early times after transplantation. This increase in CD55 expression is likely a consequence of the increase in HO-1 expression upon MLN4924 treatment, as shown in other models. These factors are also known to be implicated in the accommodation phenomenon (EC resistance to antibody aggression), for which MLN4924 could also be of interest.

[0215] Finally, the inventors found that MLN4924 preconditioning was able to block the induction of the prothrombotic molecules Tissue Factor (F3) and PAI-1 (SERPINE1), probably again through inhibition of the mTOR and NF-kB pathways, and subsequently inhibited adhesion of platelets to ECs, which suggests that this treatment could decrease the thrombosis risk following transplantation. This could have interesting implications for the preconditioning of pancreatic transplants notably. Indeed, the pancreas is extremely sensitive to ischemia-reperfusion injuries, and early pancreatic allograft failure is mainly due to thromboses that occur in 10% of recipients and are favored by prolonged ischemic times.

[0216] Altogether, the inventors identifies MLN4924 as a candidate for graft preconditioning. MLN4924 has already reached phase 3 clinical trials for patients with myeloid malignancies, suggesting low toxicity of this drug. Importantly, its use in an ex vivo perfusion setting will ensure that only the graft is targeted, with limited “side effects” and lower toxicity.

[0217] One study has evaluated the effect of neddylation blockade in lipopolysaccharide (LPS)-induced kidney damage and has showed that MLN4924 treatment of a proximal tubular cell line could inhibit the release of inflammatory cytokines by these cells upon LPS stimulation. MLN4924 treatment at low doses was also protective against LPS-induced acute kidney injury in mice. This suggests that MLN4924 preconditioning might also have protective effects on tubular cells against ischemia-reperfusion injuries in the transplant setting.

[0218] Example 3:Effect of other neddylation inhibitors on endothelial cell preconditioning

[0219] The inventors also compared the effects of two neddylation inhibitors with those of MLN4924 on endothelial cell responses:

[0220] · NAcM-OPT: a UBE2M inhibitor that inhibits the neddylation of cullins 1 to 4 and non-cullin proteins.

[0221] · HA-9104: a UBE2F inhibitor that inhibits the neddylation of cullin 5.

[0222] Primary cultures of human dermal microvascular endothelial cells (HDMECs) were incubated for 3 hours at 4°C in the presence or absence of 10 to 100 µM NAcM-OPT or HA-9104, or 10 µM MLN4924. The cells were then washed with PBS and cultured in complete culture medium at 37°C, with or without 100 U / mL TNFα and 1000 U / mL IFNγ (mimicking the inflammatory phenomena of reperfusion), for a further 4 hours. The expression of genes encoding adhesion molecules (ICAM1, VCAM1, E-SELECTIN), pro-inflammatory cytokines and chemokines (CCL2, CCL5, CX3CL1, IL6), pro-thrombotic molecules (Tissue factor) and cytoprotective molecules (HMOX1, CD55) was analysed by qPCR.

[0223] The results obtained () show that preconditioning endothelial cells with high-dose NAcM-OPT also inhibits the induction of ICAM1, VCAM1, E-selectin, CCL2, CCL5, CX3CL1 and tissue factor expression and increases HMOX1 expression, but rarely as strongly as MLN4924. HA-9104, on the other hand, reduces the expression of ICAM1, CCL5, CX3CL1, IL6 and tissue factor at high doses, but also with weaker effects than MLN4924. The combined use of NAcM-OPT and HA-9104 could potentially produce effects similar to those of MLN4924.

[0224] Therefore, the inventors demonstrated that different neddylation pathway inhibitors can be used for preservation of tissues according to the present invention.

Claims

1.A method for in vitro or ex vivo preservation of a tissue or an organ previously obtained from a mammal, for transplant of said tissue or organ in a recipient compatible mammal, the method comprisingcontacting, in vitro or ex vivo, said tissue or organ with a treating composition, the treating composition comprising a neddylation pathway inhibitor, for 1 to 12 hours, in order to obtain a treated tissue or organsaid composition comprising from 1 to 100 µM of said neddylation pathway inhibitor;washing the treated tissue or organ with a conservation composition for removing the treating composition from the treated tissue or organ, in order to obtain a washed tissue or organ; andcontacting washed tissue or organ with a preservation composition, in order to obtain a ready to be transplanted tissue or organ.2.The method according to claim 1, wherein the neddylation pathway inhibitor is one of the following compound: HA-1141, HA-9104, arctigenin, NAcM-OPT, NAcM-COV, DI-404, DI-1548, DI-1859, WS-383, DC-2, DN-2, SK-464, and, MLN4924.3.The method according to claim 1, wherein the neddylation pathway inhibitor is an inhibitor of the NEDD8-activating enzyme.4.The method according to anyone of claims 1 to 3, wherein the neddylation pathway inhibitor is MLN4924, of formula .5.The method according to anyone of claims 1 to 4, wherein the preservation compositions are IGL-1®, CELSIOR® or UW SPS-1® solutions for a contact in static cold storage, and UW MPS® or Perfadex® for a contact in hypothermic machine perfusion.6.The method according to anyone of claims 1 to 5, wherein the step of contacting washed tissue or organ with a preservation composition is carried out at a temperature comprised from 2°C to 10°C, preferably at 4°C.7.The method according to anyone of claims 1 to 6, wherein the contacting, in vitro or ex vivo, of said tissue or organ with the treating composition is carried out by flushing the treating composition by hypothermic machine perfusion, or by immersing said tissue or organ in the treating composition after manual flushing.8.The method according to anyone of claims 1 to 7, wherein the contacting, in vitro or ex vivo, of said tissue or organ with the treating composition is carried out at a temperature from 4°C to 37°C.9.Use of a neddylation pathway inhibitor, said neddylation pathway inhibitor being one of the following compunds: HA-1141, HA-9104, arctigenin, NAcM-OPT, NAcM-COV, DI-404, DI-1548, DI-1859, WS-383, DC-2, DN-2, SK-464, and, MLN4924,for in vitro preservation of a tissue or an organ previously obtained from a mammal.10.The use according to claim 9, wherein the compound is the neddylation pathway inhibitor is MLN4924, of formula .11.The use according to claim 9 or 10, wherein said compound is used at a concentration from 1 to 100 µM.

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