Use of HDL in preventing graft-versus-host disease

Abstract

The present invention relates to high density lipoprotein (HDL) or HDL mimetics for use in the prevention and / or treatment of graft-versus-host disease (GvHD) or cytokine release syndrome in a subject.

Description

[Technical Field]

[0001] The present invention relates to the prevention and / or treatment of graft-versus-host disease. [Background technology]

[0002] The therapeutic efficacy of bone marrow transplantation (BMT) is significantly compromised by graft-versus-host disease (GvHD), which is associated with significant morbidity and mortality.

[0003] GvHD is a condition for which corticosteroid therapy remains the first-line treatment. However, corticosteroid therapy is associated with numerous iatrogenic complications (e.g., diabetes, metabolic bone disorders with a risk of avascular necrosis, arterial hypertension, and dyslipidemia) and poor prognosis in patients resistant to this treatment. There is no consensus regarding the treatment of these severe cases, and immunosuppressive approaches that disrupt the T lymphocyte cell cycle remain the only available tool.

[0004] Therefore, there is a great need for alternative treatments for GvHD that avoid such side effects.

[0005] The present invention seeks to meet this need.

[0006] Donor T cell subgroups are recognized as key mediators and effectors of acute GvHD. Interactions between donor and host T lymphocytes and antigen-presenting cells (APCs) are necessary to achieve the "activated alloreactive T cell" state, which generates cytotoxic attacks against target organs. However, prior activation of APCs by exogenous or endogenous alarm signals from damaged or "distressed" cells is crucial for effective T lymphocyte recruitment and activation. Exogenous signals are a very widespread group of natural microbial patterns known as "pathogen-associated molecular patterns" (PAMPs), which are thought to be translocated from the microbiota when the body's physical barriers, particularly the intestinal mucosa, are weakened. Among these PAMPs, lipopolysaccharide (LPS) is the one most frequently studied in relation to the pathophysiology of GvHD. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Cooke et al. (2001) J. Clin. Invest. 107:1581 [Non-patent document 2] Tardif et al. (2007) JAMA 297:1675-1682 [Non-patent document 3] Diditechenko et al. (2013) Arterioscler. Thromb. Vasc. Biol. 33 :2202~211 [Non-patent document 4] Tardif et al. (2014) Eur. Heart J. 35:3277-3286 [Non-patent document 5] Di Bartolo et al. (2011) Lipids Health 10:224 pages Summary of the Invention [Problem to be solved by the invention]

[0008] We identified an increase in free active plasma LPS after allogeneic transplantation and subsequently investigated its metabolism to identify key steps altered during allogeneic bone marrow transplantation. In healthy individuals, LPS is generally transported by HDL to the liver, where it is eliminated via bile. Various plasma transporters are involved in LPS loading onto HDL. Here, we showed that a decline in circulating HDL is the limiting factor explaining the increase in free active LPS. [Means for solving the problem]

[0009] The present invention arose from the inventors' unexpected discovery that repeated administration of HDL lipoproteins neutralizes LPS in GvHD, reduces APC maturation and activation of T Tc1 lymphocytes, the effector cells of GvHD, and therefore reduces the occurrence and severity of acute GvHD.

[0010] Thus, the treatment proposed here by the inventors - HDL administration - makes it possible for the first time to prevent and treat GvHD relying on the modulation of lipid metabolism rather than toxic immunosuppressive treatment, without affecting the quality of immune reconstitution after transplantation and with minimal expected toxicity to other organs.

[0011] This is especially true in the liver, where we have demonstrated that CD8 + There was a reduction in T cell infiltration and limited activation of resident and non-resident macrophages.

[0012] Cooke et al. (2001) J. Clin. Invest. 107:1581 studied the use of a competitive LPS inhibitor, compound B975, in a mouse bone marrow transplant model. Unlike compound B975, HDL is not a competitive LPS inhibitor; rather, it neutralizes and eliminates LPS.

[0013] Furthermore, HDL has anti-inflammatory effects independent of LPS, making it of further interest in the prevention and / or treatment of GvHD, whose pathophysiology is characterized by an important inflammatory component.

[0014] Accordingly, the present invention relates to high density lipoprotein (HDL) or HDL mimetics for use in the prevention and / or treatment of graft-versus-host disease (GvHD) or cytokine release syndrome in a subject. [Brief explanation of the drawings]

[0015] [Figure 1] See Examples. [Figure 2] See Examples. [Figure 3] See Examples. [Figure 4] See Examples. [Figure 5] See Examples. [Figure 6] See Examples. [Figure 7] See Examples. [Figure 8] See Examples. [Figure 9] See Examples. [Figure 10] See Examples. [Figure 11] See Examples. [Figure 12] See Examples. [Figure 13] See Examples. [Figure 14] See Examples. [Figure 15] See Examples. [Figure 16] See Examples. [Figure 17] See Examples. [Figure 18] See Examples. [Figure 19] See Examples. [Figure 20] See Examples. [Figure 21] See Examples. [Figure 22]See Examples. [Figure 23] See Examples. [Figure 24] See Examples. [Figure 25] See Examples. DETAILED DESCRIPTION OF THE INVENTION

[0016] HDL and HDL mimics As used herein, "high density lipoprotein (HDL)" refers to the smallest and densest group of lipoprotein particles.

[0017] HDL is well known to those skilled in the art and is typically a cholesterol- and phospholipid-rich lipoprotein containing apolipoproteins A1, A-II, A-IV, CI, C-II, C-III, and E, with a density between 1.063 and 1.210 and a diameter ranging from 5 to 12 nm.

[0018] Typically, HDL used in the context of the present invention is isolated from the blood of healthy donors, in particular from healthy donor plasma, for example by ultracentrifugation-based separation techniques.

[0019] Alternatively, the HDL used in the present invention is reconstituted from purified or recombinant apolipoprotein A1, as defined below, and from selected lipids, particularly soybean phospholipids.

[0020] Thus, examples of HDL that can be used in the present invention include CSL111 as described in Tardif et al. (2007) JAMA 297:1675-1682, CSL112 as described in Diditechenko et al. (2013) Arterioscler. Thromb. Vasc. Biol. 33:2202-211, CER-001 as described in Tardif et al. (2014) Eur. Heart J. 35:3277-3286, ETC-216 (ApoA1 Milano Also known as MDCO-216, HDL reconstituted from

[0021] As used herein, "HDL mimetic" refers to a molecule that mimics the function of HDL.

[0022] Examples of HDL mimetics include gold nanoparticles coated with phospholipids and ApoA1 or ApoA1 mimetics, and HDL reconstituted from ApoA1 mimetics such as the molecule ETC-642 described in Di Bartolo et al. (2011) Lipids Health 10:224.

[0023] Medical Applications The present invention relates to high density lipoprotein (HDL) as defined in the HDL and HDL mimetics section above or an HDL mimetic as defined in the HDL and HDL mimetics section above for use in the prevention and / or treatment of graft-versus-host disease (GvHD) or cytokine release syndrome in a subject.

[0024] The present invention also relates to the use of high density lipoprotein (HDL) as defined in the HDL and HDL mimetics section above or an HDL mimetic as defined in the HDL and HDL mimetics section above for the manufacture of a medicament for the prevention and / or treatment of graft-versus-host disease (GvHD) or cytokine release syndrome in a subject.

[0025] The present invention further relates to a method for the prevention and / or treatment of GvHD or cytokine release syndrome, comprising the administration of a therapeutically effective amount of HDL as defined in the HDL and HDL mimics section above, or an HDL mimic as defined in the HDL and HDL mimics section above, in a subject in need thereof.

[0026] As used herein, "treatment," "treating," and the like refer to achieving, in whole or in part, one or more of the following results: a partial or total reduction in the extent of the disease, an improvement in clinical symptoms or indicators related to the disease, inhibiting or preventing the progression of the disease, or delaying, inhibiting, or preventing the occurrence of recurrence of the disease, in whole or in part.

[0027] In the context of the present invention, "prophylaxis," "prevention," and the like refer to any indication for successfully protecting a subject or patient (e.g., a subject or patient at risk of developing a disease) from developing, contracting, or having a disease, including the prevention of one or more symptoms of the disease.

[0028] "Subject" refers to a mammal, preferably a human. Preferably, the subject to be treated in the context of the present invention has cancer, particularly a hematological malignancy (e.g., leukemia, lymphoma, or myeloma), or a non-malignant hematological disorder such as primary immunodeficiency, medullary dysplasia, or myelodysplasia.

[0029] "Graft-versus-host disease," "GvHD," "GvH," or "graft-versus-host reaction" refers to any T-cell-mediated immune response in which donor lymphocytes react with host antigens, typically following bone marrow transplantation or bone marrow allotransplantation.

[0030] GvHD is described as "acute" or "chronic" depending on the time it takes to first occur and the type of clinical symptoms, including signs of inflammation or fibrosis, among others. One form, known as the "overlap syndrome," refers to GvHD that includes features of both acute and chronic GvHD. Acute GvHD can vary in severity between mild and very severe, while the severity of chronic GvHD is usually defined as "limited" or "extensive."

[0031] Acute GvHD most often occurs within the first 100 days after allogeneic transplantation. It usually affects the skin, liver, and intestines, but can also affect other organs. Acute GvHD may be classified according to the severity of its symptoms: - Grade 1: Mild symptoms - Grade 2: Moderate symptoms, - Grade 3: severe symptoms, - Grade 4: Very severe symptoms.

[0032] Symptoms of acute GvHD typically include burning and redness of the skin on the palms or soles of the feet, a skin rash, blisters and scaling that may spread over the body, loss of appetite, nausea, vomiting, abdominal pain, malabsorptive diarrhea, hepatocellular lysis associated with jaundice, and liver abnormalities leading to hepatocellular failure.

[0033] Chronic GvHD classically occurs three months after allogeneic transplantation. It can last for several months or the patient's lifetime. Chronic GvHD can occur immediately after acute GvHD or an asymptomatic period. Symptoms of chronic GvHD typically include skin disorders such as dryness, skin rash, itching, scaling, poikiloderma, and loss of skin elasticity up to and including more or less extensive scleroderma. Dryness of the mucous membranes and / or eyes is commonly present, resulting in a burning / foreign body sensation in the eyes and xerostomia (dry mouth), which may or may not be associated with oral lichen planus and / or ulcers. In the gastrointestinal system, chronic GvHD manifests as anorexia, abdominal pain, diarrhea, malabsorption, and nausea / vomiting. Many other organs may be affected, such as the lungs (pulmonary fibrosis) or the musculoskeletal system (polyarthritis, spasms). It should be noted that chronic extensive GvHD is often associated with impaired immune reconstitution, exposing patients to opportunistic infectious complications.

[0034] In one particular embodiment, the GvHD to be prevented or treated in the context of the present invention is acute GvHD.

[0035] In one particularly preferred embodiment, the GvHD to be prevented or treated in the context of the present invention is acute hepatic GvHD.

[0036] As mentioned above, GvHD typically occurs after bone marrow transplantation.

[0037] Thus, in one particular embodiment, the subject to be treated has undergone / will undergo a bone marrow transplant, particularly an allogeneic transplant, to prevent or treat GvHD, particularly acute GvHD.

[0038] More specifically, in one particular embodiment seeking to prevent GvHD, the subject being treated is scheduled to undergo a bone marrow transplant, particularly an allogeneic transplant.

[0039] In an alternative embodiment for treating GvHD, the subject to be treated has undergone a bone marrow transplant, particularly an allogeneic transplant.

[0040] In certain embodiments, the subject being treated has impaired digestive permeability, which may typically result from pre-transplant treatments such as chemotherapy and / or radiation therapy. Without wishing to be bound by theory, these treatments may induce apoptosis of intestinal epithelial cells, promote immune cell infiltration, and disrupt crypt / villus structure, affecting the integrity of the intestinal barrier.

[0041] As used herein, "cytokine release syndrome (CRS)" refers to a systemic inflammatory response that can be triggered by various factors, such as infections and certain drug therapies. CRS is therefore typically observed as a side effect following the administration of antibody therapy, non-protein anticancer therapy, CAR-T lymphocytes, and bone marrow allogeneic transplantation or GvHD. This syndrome is typically characterized by fever, chills, tremors, urticaria, bronchospasm with angioedema, and severe respiratory distress accompanied by hypoxia, which can lead to acute respiratory failure and death.

[0042] Thus, in certain embodiments seeking to prevent and / or treat CRS, the subject to be treated has been / is being treated with an anti-cancer agent, particularly an anti-cancer antibody, or CAR-T cells, or has undergone / will undergo a bone marrow transplant.

[0043] In a preferred embodiment for preventing and / or treating CRT, the subject being treated is / was treated with CAR-T cells.

[0044] HDL, as defined in the "HDL and HDL Mimetics" section above, or HDL mimetics, as defined in the "HDL and HDL Mimetics" section above, is typically administered to a subject in need thereof in a therapeutically effective amount.

[0045] As used herein, a "therapeutically effective amount" refers to the amount of active ingredient sufficient to eradicate, modify, control, or eliminate a disease. A "therapeutically effective amount" also refers to the amount of active ingredient that slows or minimizes the extent of a disease. It also refers to the amount of active ingredient that provides a therapeutic benefit in the treatment or management of a condition. Finally, a "therapeutically effective amount" refers to the amount of active ingredient, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease, including amelioration of symptoms associated with the disease.

[0046] The therapeutically effective amount will, of course, depend on the active ingredient in question, the mode of administration, the therapeutic indication, and the age and health of the patient.

[0047] Advantageously, the active ingredient used in the present invention is in the form of a pharmaceutical composition, optionally further comprising a pharmaceutically acceptable excipient.

[0048] As used herein, "pharmaceutically acceptable" refers to compositions and molecular entities that do not cause side effects, allergic reactions, or other adverse effects when administered to a subject. Thus, a pharmaceutically acceptable excipient or vehicle is an encapsulating agent, diluent, support, or any other non-toxic liquid, semi-solid, or solid formulating agent.

[0049] Pharmaceutical compositions used in the present invention are typically prepared to suit their mode of administration. Acceptable pharmaceutical excipients are typically determined in part by the composition being administered, as well as the particular technique used to administer the composition.

[0050] The dosage of the compound to be administered depends on each individual case and must be adapted to individual circumstances to obtain a therapeutically effective amount and optimal effect, as is well known to those skilled in the art.The therapeutically effective dose is specific to each patient and depends on various factors, including, in particular, the disorder to be treated and its severity, the activity of the specific compound used, the specific composition used, the patient's age, weight, general health, sex and diet, the administration time, administration route and excretion rate of the specific compound used, the duration of treatment, the drugs used in combination with the specific compound used, and similar factors well known in the medical field.For example, it is well known to those skilled in the art to start with a compound at a dosage level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0051] The daily dose may be administered in a single dose, or if larger amounts are administered, may be divided into several doses.

[0052] The dosage of the active ingredient depends, inter alia, on the mode of administration and can be readily determined by one skilled in the art. A therapeutically effective amount (unit dose) of the compound can be 0.01 to 500 mg / kg, preferably 0.1 to 500 mg / kg, more preferably 0.1 to 250 mg / kg, more preferably 0.1 to 100 mg / kg, more preferably 0.1 to 50 mg / kg, and even more preferably 1 to 20 mg / kg, administered once or more weekly for several weeks or months. An effective unit dose can therefore be readily determined from the calculated dose for an "average" patient weighing 70 kg.

[0053] Typically, HDL as defined in the "HDL and HDL Mimetics" section above, or HDL mimetics as defined in the "HDL and HDL Mimetics" section above, is administered to a subject in need thereof at a dose of 20 mg / kg.

[0054] In certain embodiments, HDL as defined in the "HDL and HDL Mimetics" section above, or HDL mimetics as defined in the "HDL and HDL Mimetics" section above, is administered repeatedly, preferably daily, to a subject in need thereof.

[0055] In certain embodiments, if the subject being treated is scheduled to undergo a bone marrow transplant, HDL as defined in the "HDL and HDL Mimetics" section above, or an HDL mimetic as defined in the "HDL and HDL Mimetics" section above, is administered prior to BMT, preferably during the conditioning phase, typically 7 days prior to BMT.

[0056] Preferably, HDL as defined in the HDL and HDL mimetics section above, or HDL mimetics as defined in the HDL and HDL mimetics section above, is administered prior to BMT as defined above and again after BMT, typically 1-3 days after BMT, preferably 1 day after BMT, and more preferably repeatedly between 1-24 days after BMT.

[0057] Alternatively, administration of HDL as defined in the "HDL and HDL Mimetics" section above, or HDL mimetics as defined in the "HDL and HDL Mimetics" section above, is initiated after BMT, typically 1 to 7 days after BMT, typically 1 day after BMT, and preferably repeated between 1 and 24 days after BMT.

[0058] The compositions used in the present invention may be solid, liquid, or semi-solid and may be adapted for a variety of routes of administration, including oral, rectal, nasal, ocular, topical (e.g., topical, transdermal, buccal, vaginal, or sublingual), or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intradermal).

[0059] Preferably, the HDL or HDL mimetic is administered intravenously.

[0060] Intravenous formulations comprise the active ingredient dissolved, suspended, or emulsified in a sterile vehicle, optionally in the presence of emulsifiers, stabilizers, buffers, and other conventional additives; they are usually distributed in flasks or bottles and may be air-dried in dry form for reconstitution with water or a suitable vehicle before use.

[0061] Solid pharmaceutical compositions may be tablets, capsules, powders, granules, pills, powders for reconstitution, etc. They may contain excipients such as binders, fillers, diluents, compression agents, lubricants, surfactants, colorants, flavoring agents, and wetting agents. Tablets can be coated by methods well known in the art. Suitable fillers include cellulose, mannitol, lactose, etc.

[0062] Liquid compositions for oral administration may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or they may be in a dry form to be reconstituted with water or a suitable vehicle before use. They may contain conventional additives, such as suspending agents such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, or hydrogenated edible fats, emulsifiers such as lecithin, sorbitan monooleate, or acacia gum; non-aqueous carriers (which may contain edible oils) such as almond oil, walnut oil, or fractionated coconut oil; oily esters such as glycerin, propylene glycol, or ethyl alcohol esters; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid, or optionally conventional flavorings or colorings.

[0063] In this application, the term "comprise" is intended to be inclusive of all features specifically mentioned, and optionally, to include certain additional features not specified. Furthermore, use of the term "comprising" is intended to describe embodiments in which there are no other characteristics than those specifically mentioned (i.e., "consisting of").

[0064] The invention is explained in more detail in the following figures and examples. [Example]

[0065] Example 1 In this example, we show that intravenous administration of allogeneic T lymphocytes induces early translocation of LPS as well as a low degree of neutralization of its inflammatory properties, which is mostly found in the free form in the plasma after BMT.

[0066] 5 × 10 from BALB / c (syngeneic) or C57Bl / 6 (allogeneic) donors 6 T-lymphocyte-depleted bone marrow and 1 × 10 6 In an established mouse model of GvHD consisting of lethally irradiated (8.5 Gy) BALB / c recipient mice transplanted with splenic T lymphocytes, 3-hydroxymyristic acid (3HM)—the most common hydroxylated fatty acid found in the A lipid of LPS—was quantified in the plasma and bile of recipient mice 3 and 6 days after BMT using Endoquant® technology, based on HPLC coupled with tandem mass spectrometry (Plasma: n = 26–32 mice / group in six independent experiments; Kruskal-Wallis and Dunn's post hoc tests, *: p < 0.05, **: p < 0.01, ****: p < 0.0001; Bile: n = 26–21 mice / group in three independent experiments; after one-way ANOVA and Bonferroni test, *: p < 0.05, **: p < 0.01, ****: p < 0.0001) (Figure 1).

[0067] We found that this increase in plasma LPS after bone marrow allotransplantation was primarily due to its free form. We confirmed the increase in LPS after allotransplantation using the LAL test (Limulus Amebocyte Lysate, based on the clotting of lysate from amebocytes contained in Limulus blood only in the presence of active, i.e., free, LPS) (n = 9 mice / group in three independent experiments) (Figure 2, left). This was confirmed by separating free LPS (not bound to circulating lipoproteins) from HDL-bound forms by ultracentrifugation followed by quantification using Endoquant® technology (n = 3–4 mice / group in one experiment; two-way ANOVA post-hoc test and Bonferroni test; ***: p < 0.001) (Figure 2, right).

[0068] We found that the soluble form of CD14 (sCD14) was increased in the plasma of allorecipient mice on day 15 after transplantation (n = 6–15 mice / group in three independent experiments, Kruskal-Wallis post-hoc test and Dunn's test, **: p < 0.01) (Figure 3).

[0069] The ability of recipient mouse plasma to neutralize the activity of a known concentration of LPS was studied in vitro. Briefly, HEK-Blue TLR4 / CD14 / MD-2 reporter cells were cultured in the presence of 0.5% plasma and 0.01 EU / mL standard LPS. We demonstrated that on day 3 after transplantation, the plasma of allogeneic recipient mice neutralized the activity of commercial LPS added at a known concentration (0.01 EU / mL, InvivoGen), resulting in a 21.6% reduction compared to naive mice. On day 6, the reduction was equal to 14% (n = 11–16 mice / group in two independent experiments, **: p < 0.01 after one-way ANOVA and Bonferroni test) (Figure 4).

[0070] Example 2 In this example, we show that the reduced neutralizing capacity was likely due to altered retrotransport of LPS in allogeneic recipient mice.

[0071] Reverse LPS transport (RLT) is a metabolic pathway similar to reverse cholesterol transport, in which lipophilic substances are transported by lipoproteins (mainly HDL) to the liver for recycling or elimination in the bile.

[0072] PLTP (phospholipid transfer protein) plays a key role in RLT and can load LPS onto HDL. We showed that its activity, measured by a commercial kit, was slightly reduced by total body irradiation (FBI) (day 3 after transplantation) and administration of allogeneic T lymphocytes (day 6) (n = 18-21 mice / group in three independent experiments, one-way ANOVA and Bonferroni test, ****: p < 0.0001) (Figure 5).

[0073] Nevertheless, we demonstrated that PLTP activity is not a major effector in the context of GvHD, since the complete absence of PLTP activity did not increase the mortality rate of allografted mice. In this model, lethally irradiated (10 Gy) Pltp+ / + or Pltp- / - C57Bl / 6 mice were injected with 20 × 10 IgG from C57Bl / 6 (syngeneic) or C3H (allogeneic) donors. 6 5 x 10 bone marrow and 5 x 10 6 splenic T lymphocytes were transplanted (n = 12–17 mice / group in two independent experiments, log-rank test, ns: not significant, p = 0.5338) (Figure 6).

[0074] We demonstrated that the primary limiting factor appeared to be the availability of LPS transporters, since an early decline in plasma HDL concentrations (day 6) was observed in both mouse GvHD models. Figure 7 shows the results obtained in the C57Bl / 6→BALB / c model (n = 3–11 mice / group in two independent experiments, **: p < 0.01 after Kruskal-Wallis and Dunn's tests).

[0075] Example 3 In this example, we show that loss of apolipoprotein A1 (ApoA1) synthesis and circulating HDL increases the severity of GvHD.

[0076] To study the dominant effect of HDL on RLT in GvHD, we compared mice with and without ApoA1 gene expression (WT) or non-expression (Apoa1 tm1Unc ) Lethally irradiated (10 Gy) C57Bl / 6 mice were given 20 × 10 IgG from C57Bl / 6 (syngeneic, syngeneic) or C3H (allogeneic, allogeneic) donors. 6 5 x 10 bone marrow and 5 x 10 6 Splenic T lymphocytes were transplanted.

[0077] ApoA1 is the major apolipoprotein in HDL formation, and the inventors have demonstrated that its absence results in ApoA1 tm1Unc It was shown that IFN-γ exacerbated the decline in HDL in recipient mice (d+6), resulting in an almost complete absence of circulating HDL in plasma (0.067 g / l) (n=6 mice / group in two independent experiments, Mann-Whitney test, **: p<0.01) (Figure 8).

[0078] We demonstrated that the absence of HDL in allogeneic recipient mice exacerbated the mortality and severity of GvHD after transplantation (n = 9-19 mice / group in three independent experiments, log-rank survival test, ***: p < 0.001 and after Kruskal-Wallis and Dunn's ASC test for clinical scoring, **: p < 0.01) (Figure 9).

[0079] We showed that the lack of HDL appeared to be associated with altered LPS-neutralizing capacity, as assessed by the HEK-Blue TLR4 / CD14 / MD-2 reporter cell method (preliminary data, n = 3 mice / group per experiment) (Fig. 10).

[0080] Finally, we investigated the ApoA1 tm1Unc Recipient mice showed a reduced ability to neutralize LPS on day 6 after transplantation. Indeed, ApoA1 tm1UncRecipient mice had lower plasma 3HM concentrations (i.e., total LPS) than wild-type recipient mice (WT) (n=6 mice / group in two independent experiments, Mann-Whitney test, **: p<0.01) (Fig. 11, left). On the other hand, recipient mice in these two groups had the same amount of active LPS as measured by the LAL test (n=3 mice / group in one experiment) (Fig. 11, center). This suggests that ApoA1 tm1Unc This is reflected in a lower LPS neutralization capacity or activity index than in recipient mice (Fig. 11, right).

[0081] Example 4 In this example, we show that loss of ApoA1 and HDL synthesis accelerates dendritic cell maturation and promotes IFN-γ production by T lymphocytes T in the spleen.

[0082] Conventional dendritic cell (DC) maturation (Fig. 12) and T lymphocyte polarization (Fig. 13) were observed in mice expressing (WT) or not expressing (Apoa1) the ApoA1 gene. tm1Unc ), 20 × 10 from C57Bl / 6 (syngeneic, syngeneic) or C3H (allogeneic, allogeneic) donors 6 5 x 10 bone marrow and 5 x 10 6 splenic T lymphocytes were transplanted into lethally irradiated (10 Gy) C57Bl / 6 mice and assessed by flow cytometry at the level of the spleen 6 days after transplantation.

[0083] The present inventors have identified Apoa1 tm1Unc In the spleens of recipient mice, live DC CD3 - CD19 - CD11c + IA-IE +The absolute number of DCs from allografted mice was significantly increased (Figure 12, left). These DCs show increased expression of maturation markers (CD80 and CD86) compared to DCs from WT allografted mice, both in terms of the percentage (Figure 12, center) and absolute number (Figure 12, right) of positive cells (n = 11–12 mice / group in three independent experiments, unpaired t-test or Mann-Whitney test, *: p < 0.05, **: p < 0.01, ***: p < 0.001).

[0084] We also investigated the effect of live splenic CD3 + The absolute number of T lymphocytes is Apoa1 tm1Unc The CD3 expression level in recipients was increased (Figure 13, left). + CD4 + cells (Th1 lymphocytes) and CD3 + CD8 + The percentage of cells expressing IFN-γ among Tc1 lymphocytes is higher than that of Apoa1 lymphocytes. tm1Unc The absolute number of cells was higher in recipients (Figure 13, center), and increased after 4 hours of phorbol-myristate-acetate / ionomycin stimulation (Figure 13, right) (n=11-12 mice / group in three independent experiments, unpaired t-test or Mann-Whitney test, *: p<0.05, **: p<0.01, ***: p<0.001).

[0085] Example 5 In this example, we show that ApoA1 synthesis and loss of circulating HDL can increase T cell infiltration at the level of the liver, the target organ of GvHD, and promote IFN-γ production by these liver-infiltrating T cells.

[0086] The polarity of T lymphocytes isolated from the liver (Fig. 14) was determined by the expression of the ApoA1 gene (WT) or not (Apoa1 tm1Unc ), 20 × 10 from C57Bl / 6 (syngeneic, syngeneic) or C3H (allogeneic, allogeneic) donors 6 5 x 10 bone marrow and 5 x 10 6Lethally irradiated (10 Gy) C57Bl / 6 mice transplanted with splenic T lymphocytes were assessed by flow cytometry 6 days after transplantation.

[0087] The present inventors have demonstrated that live CD3 + The absolute number of T cells is Apoa1 tm1Unc The CD3 expression level was slightly increased in recipients (n = 5-6 mice / group per experiment, Mann-Whitney test, p = 0.0628) (Figure 14, left). + CD4 + cells (Th1 lymphocytes) and CD3 + CD8 + The percentage of cells expressing IFN-γ among Tc1 lymphocytes is higher than that of Apoa1 lymphocytes. tm1Unc The levels were higher in recipients (Fig. 14, center) and showed an increase in the absolute number of cells after 4 hours of phorbol-myristate-acetate / ionomycin stimulation (Fig. 14, right) (n=5-6 mice / group per experiment, Mann-Whitney test, *:p<0.05, **:p<0.01).

[0088] Example 6 In this example, we show that ApoA1 synthesis and loss of circulating HDL lead to an increase in hepatic macrophages, primarily resident macrophages derived from monocytes, which produce elevated levels of the proinflammatory cytokines TNF-α and IL-6.

[0089] Cytokine production by resident hepatic (Kupffer cells) and non-resident (NRM) macrophages (Figure 15) was measured in cells expressing (WT) or not expressing (Apoa1) the ApoA1 gene. tm1Unc ), 20 × 10 from C57Bl / 6 (syngeneic, syngeneic) or C3H (allogeneic, allogeneic) donors 6 5 x 10 bone marrow and 5 x 10 6 splenic T lymphocytes were transplanted into lethally irradiated (10 Gy) C57Bl / 6 mice, and the tumor response was assessed by flow cytometry 6 days after transplantation.

[0090] The present inventors have demonstrated that the absolute numbers of resident (Kupffer cells) and non-resident (NRM) liver macrophages present in the liver are significantly increased by Apoa1 tm1Unc The results showed that the F4 / 80 gene was increased in recipients (n=5-6 mice / group per experiment, Mann-Whitney test, *: p<0.05) (Figure 15, left). int CD11b high The absolute number of cells expressing TNF-α (Fig. 15, center) or IL-6 (Fig. 15, right) among non-resident macrophages (NRM) was significantly increased after 4 hours of LPS stimulation. tm1Unc The expression of these inflammatory cytokines was significantly higher in recipients (n=5-6 mice / group per experiment, Mann-Whitney test, *: p<0.05). high CD11b high Regarding cells (Kupffer cells), there is an upward trend (n=5-6 mice / group per experiment, Mann-Whitney test, p=0.0823 and p=0.0519, TNF-α and IL-6, respectively).

[0091] Example 7 In this example, we show that IV administration of HDL reduces the intensity of GvHD and neutralizes available LPS.

[0092] To test the value of prophylactic IV administration of HDL to mitigate the severity of GvHD, 5 × 10 donors were administered intravenously from BALB / c (syngeneic, syng) or C57Bl / 6 (allogeneic, allogeneic) donors. 6 1 x 10 T-lymphocyte-depleted bone marrow and 1 x 10 6 Lethally irradiated (8.5 Gy) BALB / c recipients transplanted with splenic T lymphocytes were treated by IV administration of HDL isolated from human plasma (20 mg / kg) three times a week between d-1 and d+24 after transplantation.

[0093] We showed that IV HDL administration reduced the mortality (Figure 16, left) and severity (Figure 16, right) of GvHD in allografted mice (n=19-39 mice / group in 4 independent experiments, log-rank survival test and one-way ANOVA post-hoc test and Bonferroni for ASC for clinical scoring, ****: p<0.0001).

[0094] We also showed that IV administration of isolated HDL restored circulating HDL levels on day 6 (n=10-11 mice / group in three independent experiments, Mann-Whitney test, *: p<0.05) (Figure 17).

[0095] Finally, we showed that IV HDL administration appeared to reduce 3HM levels in the plasma and bile of allografted mice (n = 9-13 mice / group in four independent experiments, Mann-Whitney test). Plasma (Figure 18, left) and bile (Figure 18, center) 3HM levels were strongly correlated with circulating HDL levels (n = 13 mice / group in four independent experiments, Mann-Whitney test; n = 26 pairs, unparameterized Spearman correlation test; and n = 9-10 mice / group in four independent experiments, Mann-Whitney test; n = 19 pairs, unparameterized Spearman correlation test, ****: p < 0.0001). The 3HM concentration detected by Endoquant® technology represents the total amount of LPS present in biological fluids. IV HDL administration also appears to reduce the endotoxic activity of circulating LPS as measured by LAL (n = 7-9 mice / group in three independent experiments) (Figure 18, right).

[0096] Example 8 In this example, we present preliminary data indicating that IV HDL administration may limit the systemic inflammation associated with acute GvHD.

[0097] Preliminary data obtained by the present inventors indicate that IV administration of HDL (20 mg / kg) isolated from human plasma three times a week significantly improved survival of 5 × 10 HDL from C57Bl / 6 (allogeneic) donors. 61 x 10 T-lymphocyte-depleted bone marrow and 1 x 10 6 These results suggest that splenic T lymphocytes may reduce systemic inflammation caused by acute GvHD in lethally irradiated (8.5 Gy) BALB / c recipients.

[0098] We showed that IV HDL administration appeared to limit plasma levels of REG-3γ, an intestinal biomarker of acute GvHD, on d+15 post-transplant (n=8 mice / group per experiment) (Figure 19).

[0099] We also showed that inflammatory cytokine levels tended to decrease with IV HDL administration: interleukin-6 (IL-6) was 2-fold lower at d+6 (n=10 mice / group in two independent experiments, unpaired t-test, *: p<0.05) (Figure 20, left), and tumor necrosis factor alpha (TNF-α) was reduced by 30% at d+15 (n=3 mice / group in one experiment) (Figure 20, right).

[0100] Finally, we demonstrated that splenocytes from allografted mice treated with IV HDL administration were less able to stimulate the proliferation of allogeneic naive lymphocytes. Briefly, splenocytes from mice undergoing the C57Bl / 6 → BALB / c model were treated with mitomycin C and cultured with CFSE-marked T lymphocytes from naive C57Bl / 6 mice. The dilution of CFSE marking was analyzed by flow cytometry after 5 days of coculture. The percentage of proliferating T lymphocytes was significantly lower when cultured with splenocytes isolated from mice treated with IV HDL administration (Figure 21, left). This lower percentage appears to be due to a lower number of dividing cells (mitotic index) (Figure 21, left) and not to a difference in the rate of division (proliferation index) (Figure 21, center) (n = 6 mice / group per experiment, Mann-Whitney test, ns: not significant, **: p < 0.01).

[0101] Example 9 In this example, we present preliminary data showing that IV HDL administration limits the production of the inflammatory cytokines TNF-α and IL-12 by hepatic macrophages and the infiltration of IFN-γ-producing CD8+ T lymphocytes in the liver, as reflected in reduced hepatic GvHD histology scores, and more particularly, reduced cholangitis (inflammation of the biliary tract) in mice treated with IV HDL administration.

[0102] Preliminary data obtained by the present inventors indicate that IV administration of HDL (20 mg / kg) isolated from human plasma three times a week significantly improved survival of 5 × 10 HDL from C57Bl / 6 (allogeneic) donors. 6 1 x 10 T-lymphocyte-depleted bone marrow and 1 x 10 6 These results suggest that splenic T lymphocytes limit hepatic GvHD in lethally irradiated (8.5 Gy) BALB / c recipients.

[0103] Cytokine production by non-resident (NRM) (Fig. 22, left) and resident (Kupffer cell) hepatic macrophages (Fig. 22, center and right), and CD8 T lymphocytes (Fig. 23) was measured in 5 x 10 6 mice from C57Bl / 6 (allogeneic) donors injected with isotonic saline (+Veh) or HDL (+HDL). 6 1 x 10 T-lymphocyte-depleted bone marrow and 1 x 10 6 The T lymphocyte counts were elevated by flow cytometry in lethally irradiated (8.5 Gy) BALB / c mice transplanted with splenic T lymphocytes 6 or 24 days after transplantation.

[0104] We showed that the proportion of non-resident liver macrophages (NRMs) expressing IL-12 was decreased in HDL recipients (+HDL) 6 days after transplantation and after 4 hours of in vitro LPS stimulation (Figure 22, left) (n = 6 mice / group per experiment, Mann-Whitney test or unpaired t-test, *: p < 0.05). The proportion of resident liver macrophages (Kupffer cells) expressing TNF-α (Figure 22, left) or IL-12 (Figure 22, center) was decreased in HDL recipients (+HDL) 24 days after transplantation and after 4 hours of in vitro LPS stimulation (n = 6 mice / group per experiment, Mann-Whitney test or unpaired t-test, *: p < 0.05).

[0105] We also showed that the percentage of CD8 T lymphocytes in the livers of HDL recipient mice (+HDL) was reduced 6 days after transplantation (Fig. 23, left) (n=6 mice / group per experiment, Mann-Whitney test, *: p<0.05). These liver-infiltrating CD8 T lymphocytes synthesized less IFN-γ in HDL recipient mice (+HDL) after 4 hours of phorbol-myristate-acetate / ionomycin stimulation. IFN-γ-expressing CD3 + CD8 + The absolute number of cells (Tc1 lymphocytes) was elevated to a lower extent in mice receiving HDL injection (+HDL) than in mice receiving isotonic saline (+Veh) (Figure 23, right) (n=6 mice / group per experiment, Mann-Whitney test, *: p<0.05).

[0106] Finally, we considered the following four criteria: lobular hepatitis, inflammatory portal vein infiltration (shown in Figure 25, top; arrows indicate immune infiltration in the portal vein area; scale: 100 μm), portal vein endotheliitis, and cholangitis (shown in Figure 25, bottom; crosses indicate bile ducts; scale: 100 μm) (each parameter was scored on a scale of 0 to 3, with 0 being physiological). The histological scores of formalin-fixed, paraffin-embedded, hematoxylin / eosin-stained thin liver sections analyzed 24 days after transplantation (Figure 4) were reduced in mice treated with HDL administration (+HDL) compared with the scores of mice receiving isotonic saline (+Veh) (Figure 24, left) (n = 6-7 mice / group per experiment; Mann-Whitney test; *: p < 0.05, **: p < 0.01). The main effect of IV HDL administration was a reduction in cholangitis, i.e., inflammation of the bile ducts (Figure 24, right).

Claims

1. A composition for use in the prevention of graft-versus-host disease (GvHD) in a subject who is to undergo a hematopoietic cell transplant, for use in the treatment of graft-versus-host disease (GvHD) in a subject who has undergone a hematopoietic cell transplant, or for use in the prevention and / or treatment of cytokine release syndrome in a subject, said composition comprising high density lipoprotein (HDL) or an HDL mimetic; HDL mimics are gold nanoparticles coated with phospholipids and ApoA1 or the ApoA1 mimetics ETC-642 or CSL112, or - HDL reconstituted from the ApoA1 mimetics ETC-642 or CSL112 The composition.

2. The composition of claim 1, wherein the HDL is isolated from the blood of a healthy donor.

3. 3. The composition of claim 1 or 2, wherein the HDL or HDL mimetic is administered intravenously.

4. The composition of claim 1, wherein the HDL(mimetic) is administered repeatedly.

5. The composition of claim 1, wherein the GvHD is acute GvHD.

6. 10. The composition of claim 1, wherein the HDL(mimetic) is administered before or after hematopoietic cell transplantation.

7. 10. The composition of claim 1 for the prevention and / or treatment of cytokine release syndrome, wherein the subject is treated with chimeric antigen receptor T lymphocytes (CAR-T).

8. 5. The composition of claim 4, wherein the HDL(mimetic) is administered every two days.

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