HK1213193B - Protection of the vascular endothelium from immunologically mediated cytotoxic reactions with human cd34-negative progenitor cells - Google Patents

Description

Human CD 34-negative progenitor cells protect the vascular endothelial cell layer from immune-mediated cytotoxic reactions

Technical Field

The present invention relates to human CD 34-negative progenitor cells for medical use in the treatment of clinical conditions.

Background

Adult CD 34-negative progenitor cells are pluripotent cells with the ability to self-renew and multilineage into a variety of tissues of the hematopoietic, endothelial and mesenchymal variety. CD 34-negative progenitor cells have been associated with immunoregulatory and regenerative potential, which has been expected to have utility as cellular therapeutics for the treatment of human diseases.

For example, international patent application WO 2008/150368Al discloses the medical use of non-genetically modified CD34 negative stem cells in the treatment of the following conditions: gastrointestinal discomfort, diabetes, muscular dystrophy, and acute wound healing in surgery or physical trauma. Singer and Caplan describe the proposed mechanism of action of human Mesenchymal Stem Cells in Inflammation (N.G.Singer and A.I.Caplan: "Mesenchymal Stem Cells: Mechanisms of Inflammation", Annual Review of Pathology: Mechanisms of Disease, 2011, 457-478). Tolar et al reviewed disputes and recent opinions about MSC biology, regulation of allogenic responses by MSCs in preclinical models, and the clinical experience of MSC injections (J.Tolar, K.Le Blanc, A.Keting, B.R.Blazar: "Consise Review: Hitting the Right Spot with mesenchymal Stem Cells, Stem Cells 2010 (Stem Cells 2010), 28, 1446-. Roemering-van Rhijn et al provided results from preclinical and clinical MSC studies in Solid Organ Transplantation (m.roemering-van Rhijn, w.weimar, m.j.hoogdui: "sensory stem cells: Application for Solid Organ Transplantation", Current Opinion in Organ Transplantation (latest view of Organ Transplantation), 2012, 17, 55-62). Similarly, Hoogdujn et al evaluated the progress of Mesenchymal Stem Cell therapy in clinical Organ Transplantation (M.J.Hoogdujn, F.C.Popp, A.Grohnert, M.J.crop, M.van Rhijn, A.T.Rowshani, E.Eggenhofer, P.Renner, M.E.Reinders, T.J.Rabelink, L.J.van der lan, F.J.Dor, J.Ijzermans, P.G.Generver, C.Lange, A.Dubacrh, J.Houtgraf, B.Christ, M.Seifert, M.Shagidunn, V.Doncher, R.Denner, O.Ringke, N.Perigoo, N.Mitsutrizijn, M.Churin, M.Seift, M.S.S. Shangiden, V.Doncher, R.Denner, O.Mitsugafur, N.Mitsugazine, N.S.M.S.S.P.P.C.C.J.Patch.C.C.H.C.J.R.J.P.R.J.J.R.J.P.P.J.J.Patent, C.C.C.C.C.C.C.C.C.C.R.R.J.P.C.P.C.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R.R. LeBlanc and colleagues investigated whether Mesenchymal Stem Cells could alleviate Graft-versus-Host Disease (GvHD) following hematopoietic Stem cell transplantation (K.Le Blanc, F.Frassoni, L.ball, F.Locatelli, H.Roelofs, I.Lewis, E.Lanino, B.Sundberg, M.E.Bernardo, M.Remberger, G.Dini, R.M.Egeler, A.Baccaraupo, W.Fibbe, O.Ringden, development Committee of the European Group for Blood and bone marrow transplantation development: "sensory Stem Cells for Sterost, Resver, ace-Stem-Host-Disease, St-Host-Graft: 371), and secondary Stem cell therapy for Sterost, St-Graft-versus-Graft, 9, St.A.9 for acute Graft-versus-Graft (St. 9, St. Secondary Graft-versus-Graft; St. 9, St. Gray-Graft-versus-Graft; St. Ware. for acute Graft-versus-Graft, St. A, St. for example, St. Pati and colleagues suggested that MSC can target the treatment of vascular permeability and inflammation by local and systemic effects in the lung induced by hemorrhagic shock (S.Pati, M.H.Gerber, T.D.Menge, K.A.Wataha, Y.ZHao, J.A.Baumgartner, J.ZHao, P.A.Letourneau, M.P.Huby, L.A.Baer, J.R.Salsbury, R.A.Kozar, C.A.Wade, P.A.Walker, P.K.dash, C.S.Cox Jr, M.F.Doursout, J.B.Holcomb: "Bone white derived cultured cell in vascular endothelial cell proliferation and proliferation in the epithelial layer of mesenchymal stem cells (71) and suppress the inflammatory stem cells in the lung after hemorrhagic shock, 71% of blood shock). Charbord reviews a number of characteristics of mesenchymal stem cells including stromal and immunoregulatory capacity, which may be responsible for the versatility of injured tissue repair mechanisms (p. Charbord: "Bone marrowmesenchymal stem cells: historical overview and concept"), Human Gene therapy (Human Gene therapy), 2010, 21, 1045-56).

With the strong need to better understand the therapeutic potential of CD 34-negative progenitors and the more hierarchical medical use of these multi-functional cells, there is also an increasing clinical interest in CD 34-negative progenitors.

Disclosure of Invention

The present invention provides a human CD34 negative progenitor cell for use in protecting the vascular endothelial cell layer from immune-mediated cytotoxic reactions in a subject at risk for or suffering from a vascular inflammatory disease.

The present invention also provides a method for producing human CD 34-negative progenitor cells for the above-mentioned use, comprising the steps of:

a) isolating a CD 34-negative progenitor cell,

b) expanding CD 34-negative progenitor cells in cell growth medium for at least 12 days,

c) CD34 negative progenitor cells were harvested.

The invention further provides an assay for determining the ability of CD 34-negative progenitor cells to protect the vascular endothelial cell layer from an immune-mediated cytotoxic response by preparing a sample comprising endothelial target cells, cytotoxic CD8+ T lymphocytes and CD 34-negative progenitor cells and a reference sample comprising endothelial target cells and cytotoxic CD8+ T lymphocytes without CD 34-negative progenitor cells, and comparing the lysis of the endothelial target cells in the sample and the reference sample.

Drawings

FIG. 1 is a block diagram showing a representative experimental design showing a method of determining the ability of CD 34-negative progenitor cells to protect the vascular endothelial cell layer from immune-mediated cytotoxic reactions.

Figure 2 shows the results of protecting endothelial cells from specific lysis by allogeneic CD8+ cytotoxic T lymphocytes by using CD34 negative progenitor cells from bone marrow mesenchymal stem/stromal cells from different donors.

Figure 3 shows that lysis of endothelial cells by allogeneic CD8+ CTLs is MHC class I restricted and is not dependent on natural killer cell or lymphokine-activated killer cell activity.

Figure 4 shows that bone marrow MSCs (BM-MSCs) specifically protect endothelial cells from lysis by allogeneic CD8+ cytotoxic T lymphocytes, whereas size-matched control cells do not show a protective effect.

Fig. 5 shows a comparison of the level of protection of endothelial cells by using CD34 negative progenitor cells derived from mesenchymal stem/stromal cells of various tissues.

Terms and definitions

In the present application, certain terms are used with the meanings explained below.

As used herein, a cell is "allogeneic" with respect to a subject if the cell or any precursor cell thereof is from another subject of the same species.

As used herein, "CD 34-negative progenitor cell" refers to a stem cell that lacks CD34 on its surface. CD 34-negative progenitor cells themselves may also give rise to or differentiate into CD 34-negative stem/stromal cells. CD 34-negative progenitor cells may include hematopoietic, endothelial and/or mesenchymal progeny. In certain preferred embodiments, "CD 34-negative progenitor cells" refer to CD 34-negative mesenchymal stem/stromal cells (MSCs); whereas hematopoietic and endothelial progenitor cells eventually begin to express CD34 and other co-existing markers during maturation.

As used herein, a "vascular endothelial cell layer" shall include, but is not limited to, cells that line the interior surface of a blood vessel. In particular, the vascular endothelial cell layer includes endothelial cells that directly contact blood.

As used herein, "immune-mediated cytotoxic response" refers to, but is not limited to, a cell-mediated immune response that results in the damage or death of a target cell, wherein the immune response acts directly on the target cell. In certain embodiments, the immune-mediated cytotoxic response comprises MHC-mediated cellular immunity.

As used herein, "CD 34-negative mesenchymal stem/stromal cells" refers to stem cells that meet the three lowest criteria recommended by the international cell therapy association: (1) plastic-attachment when maintained under standard culture conditions using tissue culture flasks; (2) expresses CD105, CD73 and CD90 and, when measured by flow cytometry, does not express CD45, CD34, CD14 or CDllb, CD79a or CD 19; (3) ability to differentiate into osteoblasts, mature adipocytes and chondroblasts under standard in vitro differentiation conditions (M.domimici, K.le Blanc, I.Mueller, I.Slaper-Cortenbach, F.Marini, D.Krause, R.deans, A.Keting, Dj.Prockop, E.Horwitz: "minimum criterion for defining multipotent mesenchymal stem cells". The International Society for Cellular Therapy positioning statement, "Cytotherapy (cell Therapy), 2006, 8, 315-7).

In certain embodiments, "CD 34 negative mesenchymal Stem/stromal cells" further refers to MSCs (PD-L1) expressing B7-H1 following stimulation with γ -IFN (M. Najar, G. Raicevic, H.F. Kazan, C.De Bruyn, D.Bron, M.Toung, L.Languaux: "Immune-related antigens, surface molecules and modulators in human mesenchymal Stem cells" expression and influence of inflammation-initiated expression and influence of Immune-related antigens, surface molecules and modulators in human mesenchymal Stem cells "m Rev.8, 1188-98; S.Ti. proteins, C.V.vary, expression and influence of inflammation-initiated expression and influence of Immune-related antigens, surface molecules and modulators in human mesenchymal Stem cells" m Rev.8, 1188-98; S.Ti., C.V.V.S.V.S.5, S.V.S.S.J.S.J.S.S.J.S.S.I.S.S.I.I.S.I.I.S.S.I.S.I.I.I.S.I.I.I.I.S.I.I.I.I.S.I.I.I.I.I.I.I.I.I.A.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.A.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I.I, jurewicz, a.augello, a.vernier, s.dada, s.la Rosa, m.selig, j.godwin, k.law, c.placidi, r.n.smith, c.capella, s.rodig, c.n.adra, m.atkinson, m.h.sayegh, r.abdi: "immunomodulating function of bone marrow-derived mesenchymal stem cells in experimental autoimmune type 1 diabetes", J Immunol.2009, 183, 993-; chang, m.l.yen, y.c.chen, c.c.chien, h.i.huang, c.h.bai, b.l.yen: "Placenta-derived pluripotent Cells exhibit enhanced immunity in the presence of interferon-gamma", Stem Cells (Stem Cells), 2006, 24, 2466-77.

Unless otherwise stated, "vascular inflammatory disease" refers to, but is not limited to, an immune response that causes inflammation of vascular tissue, including large and small arteries, veins and lymphatic vessels.

As used herein, "subject" refers to any animal, such as a human, non-human primate, mouse, rat, guinea pig, or rabbit. Preferably, the subject is a human.

As used herein, "protecting the vascular endothelial cell layer" refers to slowing, stopping, or reversing the development of an immune-mediated cytotoxic response against any vascular tissue.

Detailed Description

The vascular endothelial cell layer is the main subject of various vascular inflammatory diseases. The inventors of the present invention have recognized that specific protection of the endothelial cell layer has great clinical and health economic value from the point of view of risk adaptation, personalized prevention and/or therapeutic intervention.

The inventors have conducted intensive studies on the activation state and viability of the vascular endothelial cell layer, and found that CD 34-negative progenitor cells can inhibit endothelial cell layer-specific cytotoxic responses in a pharmacologically useful and dose-dependent manner.

Thus, in a first aspect, the present invention provides a human CD34 negative progenitor cell for use in protecting the vascular endothelial cell layer from immune-mediated cytotoxic reactions in a subject at risk for or suffering from a vascular inflammatory disease.

In one embodiment of the invention, the vascular inflammatory disease comprises vascular endothelial cell layer-specific cell lysis caused by CD8+ cytotoxic T lymphocytes. The inventors demonstrated that in some cases of vascular inflammatory disease, the endothelial cell layer is a direct target for CD8+ cytotoxic T lymphocytes. The therapeutic use of CD 34-negative progenitor cells is particularly effective in interfering with CD8+ cytotoxic T lymphocyte-mediated cytotoxic responses.

In a specific embodiment of the invention, the CD8+ cytotoxic T lymphocytes comprise endothelial cell layer specific cytotoxic T lymphocytes that are CD27 negative and CD28 negative. The inventors have surprisingly found evidence of the presence of endothelial cell layer specific cytotoxic T lymphocytes that are CD27 negative and CD28 negative and do not recognize hematopoietic targets. These cells exhibit phenotypically and functionally significant characteristics. For example, their lytic activity is enhanced by CD4+/CD25+/FoxP3+ regulatory T lymphocytes (TRegs). When CD 34-negative progenitor cells were administered, the inventors achieved significant inhibition of endothelial cell lysis by this particular type of CTL.

The present invention therefore provides a layered therapeutic use of CD 34-negative progenitor cells, ensuring risk-adapted, personalized prevention and/or therapy with CD 34-negative progenitor cells. Accordingly, advantageous medical uses of CD 34-negative progenitor cells according to the invention include the clinical conditions defined below.

In one embodiment of the invention, the vascular inflammatory disease comprises an alloresponse to an intravascular endothelial cell layer of a solid organ transplant that is allogeneic with respect to the subject.

In another embodiment, the vascular inflammatory disease comprises transplantation-related complications following allogeneic hematopoietic stem cell transplantation.

For example, transplant-related complications include graft versus host disease (GvHD). In particular, graft versus host disease may be characterized by major selective damage to the gastrointestinal tract, liver, skin including mucosa, pulmonary system, and combinations thereof. In certain embodiments of the invention, graft versus host disease comprises steroid refractory acute or chronic GvHD.

Another example of a transplant related complication is microvascular disease, such as, for example, hepatic Vein Occlusion Disease (VOD), as one example.

Human CD 34-negative progenitor cells can also be used to protect the vascular endothelial cell layer in vascular inflammatory diseases, including acute, inflammatory, or allergic vasculitis due to autoimmune responses. Such uses include, but are not limited to, allergic granulomatous disease, giant cell arteritis, wegener's granulomatosis, Takawasaki disease, thromboangiitis obliterans (Buerger's disease), polyarteritis nodosa, Churg-Strauss-syndrome, microscopic polyangiitis, cryoglobulinemic vasculitis, urticaria vasculitis, eye-mouth-genital triple syndrome, goodpasture's nephritis syndrome, post-infection vasculitis, and drug-induced vasculitis.

In another embodiment, the vascular inflammatory disease comprises chronic inflammation of the vascular endothelial cell layer. For example, the chronic inflammation may include atherosclerosis. Chronic inflammation may also include vasculitis associated with rheumatoid disease.

In a preferred embodiment of the invention, the CD 34-negative progenitor cells include CD 34-negative mesenchymal stem/stromal cells. CD 34-negative progenitor cells can be selected from, for example, bone marrow, umbilical cord, placenta, and adipose tissue CD 34-negative progenitor cells and combinations thereof. In a preferred embodiment of the invention, the CD 34-negative progenitor cells are bone marrow CD 34-negative progenitor cells. Especially preferred are bone marrow CD34 negative mesenchymal stem/stromal cells.

Preferably, the CD34 negative progenitor cells are capable of reducing an immune-mediated cytotoxic response against vascular endothelial cells by at least 50% compared to endothelial cells not protected by CD34 negative progenitor cells. Suitable assays for reducing immune-mediated cytotoxic responses according to the present invention are described in further detail below.

Human CD 34-negative progenitor cells are preferred, but not only suitable for injection into the bloodstream of a subject, for example by introducing such cells by injection into one of the veins or arteries of a subject. For example, such administration may also be performed one or more times and/or over one or more extended periods of time. A single injection is preferred, but in some cases repeated injections over time may be necessary. Preferably, the CD34 negative progenitor cells are mixed with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01 to 0.1 molar, preferably 0.05 molar phosphate buffer or 0.8% physiological saline. Moreover, such pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions, with examples of non-aqueous solvents being propylene, ethylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions and suspensions include physiological saline and buffer media. Parenteral vehicles include sodium fluoride solution, ringer's dextrose, dextrose and sodium chloride, lactated ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as ringer's dextrose, ringer's dextrose-based substances, and the like. Fluids commonly used for intravenous administration are described, for example, in Remington: the science and Practice of Pharmacy, 20 th edition, page 808, Lippincott, Williams and Wilkins (2000).

Different administration regimens may be used. Preferably, the human CD 34-negative progenitor cells are suitable for injection into the bloodstream of a subject in a therapeutically effective amount. Therapeutically effective amounts may include, for example, the following ranges: l × l0²To about lxl 0⁸About lxl 0 cells per kilogram body weight³To about l0 × l0⁷About lxl 0 cells per kilogram body weight⁴To about lxl 0⁶About per kilogram of body weight of cellsl×l0⁴To about lxl 0⁵About lxl 0 cells per kilogram body weight⁵To about lxl 0⁶About 5 xl 0 cells per kilogram body weight⁴To about 0.5 xl 0⁵About lxl 0 cells per kilogram body weight³About lxl 0 cells per kilogram body weight⁴About 5 xl 0 cells per kilogram body weight⁴About lxl 0 cells per kilogram body weight⁵About 5 xl 0 cells per kilogram body weight⁵About lxl 0 cells per kilogram body weight⁶Cells per kilogram body weight and about lxl 0⁷Cells per kilogram body weight. These values for CD34 negative progenitor cells were found to be particularly therapeutically effective for transplantation.

In a variant of the invention, CD 34-negative progenitor cells are used prophylactically to protect the vascular endothelial cell layer of a subject at risk for vascular inflammatory disease. As used herein, prophylactic use shall include, but is not limited to, administration of cells to a subject who is intended to receive a solid organ transplant that is allogeneic with respect to the subject. In another example, prophylactic use comprises administering CD 34-negative progenitor cells to a subject who is intended to receive an allogeneic hematopoietic stem cell transplant. A further example of prophylactic use includes administration of CD 34-negative progenitor cells to a subject who has received an allograft, but has not yet developed an endothelial complication. The risk of vascular inflammatory disease may also include, for example, genetic disorders, autoimmune diseases, cancer, smoking, alcoholism, and combinations thereof. From the perspective of risk adaptation and personalized prevention, the inventor realizes that the targeted protection of the vascular endothelial cell layer has important clinical and health economic values.

Such risk stratification uses may include, for example, but are not limited to, prophylactic, i.e., prior, receipt of CD 34-negative progenitor cells by patients at risk of development of post-transplant endothelial complications. For example, CD 34-negative progenitor cells can be administered to a subject just prior to, during, and/or up to about six weeks before and/or after an intervention such as an allogeneic cell or organ transplant. Administration of CD 34-negative progenitor cells can also be extended to the acute and/or chronic phase of endothelial complications up to about 100 days after intervention. In addition, CD 34-negative progenitor cells can be therapeutically administered to a subject when an endothelial complication and/or a symptom characteristic thereof develops or is currently developing.

The CD 34-negative progenitor cells can be allogeneic with respect to the subject. The CD34 negative progenitor cells may also be autologous with respect to the subject. Alternatively, a combination of allogeneic and autologous CD34 negative progenitor cells may be used.

In one embodiment, the CD 34-negative progenitor cells are autologous with respect to the solid organ transplant and allogeneic with respect to the subject. In another embodiment, the CD34 negative progenitor cells are allogeneic with respect to hematopoietic stem cell transplantation and autologous with respect to the subject. Also included are situations where CD 34-negative progenitor cells are allogeneic with respect to the subject and solid organ transplantation or hematopoietic stem cell transplantation.

In one embodiment, CD 34-negative progenitor cells are used in combination with at least one additional active ingredient. For example, the at least one additional active ingredient may have a pharmacological activity selected from: anti-inflammatory activity, anti-ischemic activity, anti-thrombotic activity and combinations thereof. Additionally or alternatively, the at least one additional active component may have the ability to protect endothelial cells from allorecognition and/or lysis. In certain examples, the at least one additional active component comprises a deoxyribonucleic acid derivative, such as defibrotide.

In a second aspect, the present invention provides a method of producing human CD 34-negative progenitor cells for any one of the uses described above, the method comprising the steps of: a) isolating CD 34-negative progenitor cells; b) expanding CD 34-negative progenitor cells in cell growth medium for at least 12 days; c) CD34 negative progenitor cells were harvested.

In one embodiment, in method step a), the CD34 negative progenitor cells are isolated from a tissue comprising: bone marrow, umbilical cord, placenta, and adipose tissue, or a combination thereof. The inventors have found that progenitor cells from these tissue sources are particularly suitable for protecting the vascular endothelial cell layer in vascular inflammatory diseases. Preferably, the CD 34-negative progenitor cells are CD 34-negative mesenchymal stem/stromal cells. Most preferred are CD34 negative mesenchymal stem/stromal cells from bone marrow.

According to a further embodiment of the method, the cell growth medium of method step b) comprises a medium comprising:

human platelet lysate free of solid matter having a diameter greater than 0.22 μm, wherein the lysate represents between 2% and 15% of the total volume of the cell growth medium,

-a human Fresh Frozen Plasma (FFP) filtrate, free of solid matter having a diameter of more than 0.22 μm, wherein the FFP filtrate represents 1% to 10% of the total volume of the cell growth medium,

-heparin in a concentration of 0U/ml to 10U/ml in the cell growth medium,

-L-glutamine at a concentration of 0.5mM to 10mM, and

-a serum-free, low glucose medium suitable for mammalian cell growth, wherein the serum-free, low glucose medium comprises 75% to 97% of the total volume of the cell growth medium.

Herein, human freshly frozen plasma refers to the liquid portion of human blood that has been centrifuged, separated, and frozen to a solid within hours of collection at-18 ℃ or colder. The inventors performed the expansion of CD 34-negative progenitor cells in this medium, which protected the vascular endothelial cell layer particularly strongly from immune-mediated cytotoxic reactions. Hereinafter, this medium is referred to as "Bio-1" medium.

In a preferred embodiment, CD 34-negative progenitor cells are harvested in method step c), which grow predominantly adhering to the surface that is in contact with the cell culture medium, such as the wall of a cell culture dish or cell culture vessel.

Preferably, process steps a) to c) are carried out under Good Manufacturing Practice (GMP) and/or current good manufacturing practice (cGMP).

Using these methods, the inventors have completed the in vitro expansion and positive selection of better defined, highly transplantable cells with very high vascular endothelial cell layer protection efficacy, whereas conventional methods typically produce heterogeneous mixtures of different subpopulations of CD 34-negative progenitor cells.

In a third aspect, the invention provides a method of determining the ability of CD 34-negative progenitor cells to protect the vascular endothelial cell layer from immune-mediated cytotoxic reactions by preparing a sample comprising endothelial target cells, cytotoxic CD8+ T lymphocytes and CD 34-negative progenitor cells and a reference sample comprising endothelial target cells and CD8+ cytotoxic T lymphocytes without CD 34-negative progenitor cells, and comparing the lysis of the endothelial target cells in the sample and the reference sample.

In one embodiment, the endothelial target cells and/or CD 34-negative progenitor cells are allogeneic with respect to CD8+ T lymphocytes.

In another embodiment, CD8+ T lymphocytes are co-cultured with allogeneic endothelial cells in the presence of interleukin-2 (IL-2) prior to preparing the sample and reference sample with endothelial target cells and the CD8+ T lymphocytes. The co-culture may be maintained, for example, for at least one day, preferably at least 3 days, more preferably at least 5 days, and most preferably at least 7 days.

In another embodiment, at least one additional active component is added to the sample and/or the reference sample. For example, the at least one additional active ingredient may have a pharmacological activity selected from: anti-inflammatory activity, anti-ischemic activity, anti-thrombotic activity and combinations thereof. Additionally or alternatively, the at least one additional active component may have the ability to protect endothelial cells from allorecognition and/or lysis. In certain examples, the at least one additional active component comprises a deoxyribonucleic acid derivative, such as defibrotide.

Using this approach, the inventors completed a potency assay to assess the ability of CD 34-negative progenitor cells from different sources to protect the vascular endothelial cell layer. Furthermore, an in vitro test for positive prediction of the efficacy of CD34 negative progenitor cells in vivo was achieved.

The method may also be used to analyze the blood of a subject for the presence and/or pathophysiological activity of endothelial-cytotoxic CD8+ T lymphocytes before, during and/or after receiving a transplant and/or developing an endothelial complication.

For example, in the case of a solid organ transplant that is allogeneic with respect to the subject, the endothelial target cells can include cells derived from a solid organ donor. In another case of solid organ transplantation, which is allogeneic with respect to the subject, CD 34-negative progenitor cells can include cells derived from a solid organ donor. In certain instances where the solid organ transplant is allogeneic with respect to the subject, the endothelial target cells and CD34 negative progenitor cells may include cells derived from a solid organ donor. Additionally or alternatively, CD 34-negative progenitor cells can also include cells derived from at least one third-party donor that is not the subject or a solid organ donor.

In the case of allogeneic hematopoietic stem cell transplantation, the endothelial target cells may include cells derived from the subject, i.e., the transplant recipient. In another case of allogeneic hematopoietic stem cell transplantation, CD 34-negative progenitor cells may include cells derived from the transplant recipient. In certain cases of allogeneic hematopoietic stem cell transplantation, the endothelial target cells and CD 34-negative progenitor cells may include cells derived from the transplant recipient. Additionally or alternatively, CD 34-negative progenitor cells can also include cells derived from at least one third-party donor that is not a transplant recipient or a hematopoietic stem cell donor.

As such, the method can not only be used to determine a subject's susceptibility to developing an immune-mediated cytotoxic response based on the use of individuals who use cytotoxic CD8+ T lymphocytes derived from the subject's blood in combination with context-specific endothelial target cells identified above, but can also select CD 34-negative progenitor cells that exhibit particularly potent protection of endothelial target cells.

As a result, patients may stratify, for example, according to their risk of vascular inflammatory disease. Furthermore, personalized prevention and/or therapy can be designed.

Detailed Description

In the following examples, certain embodiments of the present invention are explained in more detail with reference to figures and experimental data. The examples and figures are not intended to limit the invention.

Example 1: experimental design cytotoxicity assays

The following example describes an experimental setup designed by the inventors to evaluate the efficacy of CD 34-negative progenitor cells in protecting the vascular endothelial cell layer from immune-mediated cytotoxic reactions.

FIG. 1 is a block diagram illustrating one representative embodiment of the cytotoxicity assays of the invention. Monocytes (PBMC, 10) from peripheral blood may be used as a source of cytotoxic cells. PBMCs may for example be derived from healthy third party donors. Alternatively, PBMCs of the subject to be treated may be used.

Next, PBMCs can be further selected for CD8+ T lymphocytes (11). Selection for CD8+ T lymphocytes may, for example, comprise enrichment of CD8+ T lymphocytes from PBMCs by immunoseparation. Immune isolation may, for example, involve back selection of CD8+ T lymphocytes by eliminating non-CD 8+ T lymphocytes. A suitable method for enriching CD8+ T lymphocytes is non-contact selection, for example using immunomagnetic microparticles. Unwanted cells can be targeted, for example, by removal of antibody complexes that recognize CD4, CD14, CD16, CD19, CD20, CD36, CD56, CD66B, CD123, TCRy/δ, glycophorin a, and dextran-coated magnetic particles. Thereafter, the phenotypic purity of the selected cells can be confirmed by, for example, flow cytometry. Preferably, the majority of the selected cells are CD8+ cytotoxic T lymphocytes (CTL, 12).

In the next phase, CD8+ CTL can be maintained co-cultured (14) with endothelial-stimulating cells allogeneic to CD8+ CTL (13). Preferably, the endothelial-stimulating cells are unable to undergo mitosis. A suitable cell line is for example the SV40 large T antigen transformed microvascular endothelial cell line CDC/EU HMEC-1 (HMEC). Co-culture of endothelial-stimulating cells and CD8+ CTL, for example, can be maintained for about seven days. Preferably, the co-culture further comprises stimulating CD8+ CTL with interleukin-2.

The preparation of endothelial target cells (17) may be subjected to a labeling step (18), wherein they are labeled with a first label, such that the endothelial target cells are distinguished from other non-target cells. For example, such labels may include fluorescent compounds that have different emission characteristics when present in the plasma membrane of a cell than when present outside of the plasma membrane. A suitable compound is, for example, 3' dioctadecyl oxacarbocyanine perchlorate (DIOC 183). The target cells may be, for example, endothelial cells from transplanted tissue. The target cell may be an endothelial cell line, such as the microvascular endothelial line CDC/eu.

In the next stage, a sample (22) is prepared by binding a first fraction of labeled target cells (20), a first fraction of effector cells (16), and CD34 negative progenitor cells (21). CD 34-negative progenitor cells can be allogeneic with respect to endothelial target cells and effector cells, for example. The ratio of endothelial target cells and CD 34-negative progenitor cells may be, for example, 5: 1. The ratio of effector cells to endothelial target cells may be, for example, 20:1, 10:1 or 5: 1. A reference sample (22') was prepared by combining a second fraction of labeled target cells (20') with a second fraction of effector cells (16') without CD34 negative progenitor cells in the same proportions as in the sample described above. Incubation of the sample and reference samples was maintained (23, 23'). The incubation may be maintained, for example, for about four hours.

Thereafter, the amount of lysed endothelial target cells in the sample and the reference sample is determined (24, 24'). For this purpose, the lysed endothelial target cells may be labeled with a second label, such that the lysed target cells are distinguished from non-lysed target cells. Such a second label may for example be a stain suitable for positive staining of dead cells. For example, fluorescent stains such as propidium iodide may be used. A suitable method for determining lysed endothelial target cells may be, for example, flow cytometry. For example, the percentage of lysed cells can be determined by flow cytometry determining the amount of cells carrying the first and second tags, e.g., DIOCI83 and PI, in the total population of cells carrying the first tag, e.g., DIOCI 83.

In addition, the percentage of target cells specifically lysed by effector cells can be corrected by subtracting the percentage of randomly lysed cells. For this purpose, a third fraction (25) of labeled target cells (19) is prepared which do not contain effector cells nor CD 34-negative progenitor cells. The third fraction (25) is further processed in the same way as the sample and the reference sample, after which the amount of randomly lysed endothelial target cells in the third fraction (26) is determined as described above.

Example 2: third party CD34 negative progenitor cells protect endothelial cells from lysis by allogeneic CD8+ CTL

The cytotoxicity assay described in example 1 was used to assess lysis of endothelial target cells (HMECs) by allogeneic cytotoxic T lymphocytes with and without third party CD 34-negative progenitor cells. In this case, CD34 negative mesenchymal stem/stromal cells isolated from bone marrow were used. BM-MSCs were expanded in Bio-1 medium, which resulted in favorable cell populations for therapeutic use from the standpoint of maintaining stem cell characteristics, cell viability, and efficacy in protecting the vascular endothelial cell layer from immune-mediated cytotoxic reactions.

Different ratios of effector cells and target cells were prepared for 20:1, 10: 1and 5: 1. For each ratio, six individual samples without bone marrow MSCs and six individual samples with bone marrow MSCs (BM-MSCs) were prepared. For the latter, target cells were incubated with a single donor of BM-MSCs for 24 hours prior to addition of effector cells.

The results of the experiment are shown in figure 2. The figure shows the specific lysis of target cells in percentage with respect to the respective effector to target cell ratio in samples without BM-MSC (diamond marker) and with BM-MSC (square marker). Data represent mean and standard deviation of six individual experiments. BM-MSCs were found to inhibit allogeneic endothelial cell lysis by CD8+ CTLs with high significance at all tested E/T ratios (; p < 0.001). By adding CD34 negative bone marrow mesenchymal stem/stromal cells, the specific lysis of allogeneic endothelial target cells was reduced by about 65% on average, i.e. 28.3 ± 5.8% to 9.7 ± 8.3%.

Example 3: immunomodulatory effects of CD 34-negative progenitor cells are specific for the endothelial cell layer

As a control, experiments were performed as described in example 2, except that CD34 negative BM-MSCs were not added to endothelial cells, but were pre-incubated with effector cells and then removed. In this case, no decrease in specific lysis of endothelial target cells was observed when effector cells were preincubated with BM-MSCs, compared to effector cells not preincubated with BM-MSCs. It can therefore be concluded that the immunomodulatory activity of BM-MSCs is involved in the specific interaction of BM-MSCs with endothelial target cells.

Example 4: endothelial target cell lysis by allogeneic CD8+ CTL is MHC class I restricted and independent Activation of killer cell activity in natural killer cells or lymphokines

As a further control, it was investigated whether the lytic activity of allogeneic CD8+ cytotoxic T lymphocyte effector cells is antigen-specific by MHC class I presentation limited to allogeneic antigens. For this purpose, endothelial target cells were subjected to CD8+ effector cells as in example 2, but in the presence of neutralizing MHC class I antibodies (W6/32). In addition, it was shown that effector cells do not have activities equivalent to natural killer cells or non-specific lymphokine-activated killer cells. This control was accomplished by performing the experiment as in example 2, but wherein for natural killer cells (K562), the endothelial target cells were replaced with the control target cell line.

The results of these control experiments are summarized in fig. 3. The columns represent the arithmetic mean and standard deviation of specific lysis of target cells at an effector cell/target ratio of 20 in four independent experiments with four individual BM-MSC donors each. The results show that the presence of neutralizing anti-MHC class I antibodies significantly reduced the specific lysis of the target cells (.;: p <0.001), confirming that the lytic activity of the effector cells is alloantigen-specific. Furthermore, lysis of the control target K562 was significantly reduced (. about.. p <0.002), indicating that cytotoxic T lymphocyte effector cells did not have activity against natural killer cells or non-specific lymphokine-activated killer cells.

Example 5: protection of endothelial target cells from lysis by CD8+ CTL Effector cells against CD34 negative progenitors Cell-specific

CD 34-negative progenitor cells such as bone marrow-derived CD 34-negative mesenchymal stem/stromal cells are relatively large cells. Therefore, it was assessed whether the protection of endothelial target cells by BM-MSC was based on steric inhibition from CD8+ T lymphocyte effector cells that access endothelial cells via BM-MSC, rather than on the immunomodulatory capacity of BM-MSC. The experimental design was similar to example 2, but in which fibroblast-like cells from human heart tissue were added to endothelial target cells instead of BM-MSCs. The size of the fibroblast-like cells was matched to the BM-MSC.

The results are shown in fig. 4. The columns represent the arithmetic mean and standard deviation of specific lysis of endothelial target cells for three independent experiments with three different BM-MSC donors each, normalized in percent to lysis of unprotected endothelial cells. No protection of endothelial target cells was observed from the addition of size-matched control cells (.: no significance), indicating that protection of endothelial target cells by BM-MSCs is immune-related and not due to steric inhibition.

Example 6: CD34 negative stem cells are immunologically poor (immunogenic negative)

It is important that the CD 34-negative progenitor cells are not themselves immunogenic. For this reason, the immunogenicity of bone marrow CD34 negative mesenchymal stem/stromal cells was investigated. The experiment was performed as in the previous example 2, but in which not endothelial cells but bone marrow mesenchymal stem/stromal cells were used as target cells without endothelial cells. No lytic activity of allogeneic CD8+ cytotoxic T lymphocyte effector cells was observed when BM-MSCs were presented as targets. This finding is particularly consistent with the poor immunity of CD 34-negative progenitor cells and mesenchymal stem/stromal cells.

Example 7: endothelial protective capacity of CD 34-progenitor cells from different sources

Comparison of the efficacy of CD 34-negative progenitor cells derived from different tissue sources in protecting endothelial target cells from CD8+ CTL-mediated lysis. The experiment was performed as in example 2, where CD 34-progenitor cells (21) were bone marrow derived MSCs (BM-MSCs, 9 separate samples), or umbilical cord derived MSCs (UC-MSCs, 4 separate samples), or amniotic membrane derived MSCs (AMC-MSCs, 3 separate samples). The results of these experiments are summarized in fig. 5. Each column pair shows the arithmetic mean and standard deviation of lysis of specific endothelial target cells with no MSC protection (filled columns) and with MSC protection (shaded columns) in percent depending on the MSC source. All MSCs tested were able to protect endothelial target cells from CD8+ CTL-mediated lysis. The most potent protection was observed with BM-MSCs, with an average reduction of 72.3% compared to endothelial cell lysis without MSC protection. AMC-MSC reduced endothelial cell lysis on average by about 53.2%, UC-MSC still reduced endothelial cell lysis on average by about 39.5%. This example also highlights the following importance of the method: the ability of CD 34-negative progenitor cells to protect the vascular endothelial cell layer from immune-mediated cytotoxic reactions was determined in order to assess the protective efficacy of cells from different sources and to positively predict the effect in vivo of CD 34-negative progenitor cells.

Claims (20)

1. Use of human CD 34-negative mesenchymal stem cells for the manufacture of a medicament for protecting the vascular endothelial cell layer of a subject at risk for or suffering from a vascular inflammatory disease from a CD8+ cytotoxic T lymphocyte-mediated cytotoxic response.

2. The use of claim 1, wherein the medicament comprises CD 34-negative progenitor cells, and the CD 34-negative progenitor cells consist of CD 34-negative mesenchymal stem cells.

3. The use of claim 2, wherein the CD 34-negative mesenchymal stem cells are administered as a mixture comprising a pharmaceutically acceptable carrier, wherein the mixture comprises CD 34-negative progenitor cells and the CD 34-negative progenitor cells consist of CD 34-negative mesenchymal stem cells.

4. The use according to claim 1, for protecting the vascular endothelial cell layer from CD8+ cytotoxic T lymphocyte-mediated cytotoxic reactions, for preventing vascular inflammatory disease in a subject at risk of developing post-transplant endothelial complications.

5. The use of claim 1, wherein the CD8+ cytotoxic T lymphocytes comprise endothelial cell specific cytotoxic T lymphocytes that are CD27 negative and CD28 negative.

6. The use of claim 1, wherein the vascular inflammatory disease comprises an alloresponse to vascular endothelial cells transplanted to a solid organ that is allogeneic to the subject.

7. The use of claim 1, wherein the vascular inflammatory disease comprises a transplant-related complication following allogeneic hematopoietic stem cell transplantation.

8. The use of claim 7, wherein the transplant-related complication comprises graft versus host disease (GvHD).

9. The use of claim 7, wherein the transplant-related complication comprises microvascular disease.

10. The use of claim 1, wherein the vascular inflammatory disease comprises acute, inflammatory, or allergic vasculitis due to an autoimmune response.

11. The use of claim 10, wherein the acute, inflammatory, or allergic vasculitis comprises allergic granulomatosis, giant cell arteritis, wegener's granulomatosis, takawasaki disease, thromboangiitis obliterans (Buerger's disease), polyarteritis nodosa, Churg-Strauss-syndrome, microscopic polyangiitis, cryoglobulinemic vasculitis, urticaria vasculitis, eye-mouth-genital triple syndrome, goodpasture's syndrome, post-infection vasculitis, or drug-induced vasculitis.

12. The use of claim 1, wherein the vascular inflammatory disease comprises chronic inflammation of the vascular endothelial cell layer.

13. The use of claim 2, wherein said CD34 negative progenitor cells are selected from the group consisting of bone marrow, umbilical cord, placenta, and adipose tissue CD34 negative progenitor cells, or a combination thereof.

14. The use of claim 2, wherein the CD 34-negative progenitor cells are characterized by expression of CD105, CD73, and CD90, and do not express CD45 and CD34, do not express CD14 or CD11b, and do not express CD79a or CD 19.

15. The use of claim 1, wherein the CD 34-negative mesenchymal stem cell is a bone marrow CD 34-negative mesenchymal stem cell.

16. The use of claim 1, wherein the CD34 negative mesenchymal stem cells are used in combination with at least one additional pharmacologically active component.

17. The use according to claim 16, wherein the at least one additional pharmacologically active component has a pharmacological activity selected from the group consisting of anti-inflammatory activity, anti-ischemic activity, anti-thrombotic activity, and combinations thereof.

18. A method of determining the ability of CD34 negative mesenchymal stem cells to protect the vascular endothelial cell layer from immune-mediated cytotoxic reactions by preparing a sample comprising endothelial target cells, cytotoxic CD8+ T lymphocytes and CD34 negative mesenchymal stem cells and a reference sample comprising endothelial target cells and cytotoxic CD8+ T lymphocytes without CD34 negative mesenchymal stem cells and comparing the lysis of endothelial target cells in the sample and the reference sample.

19. The method of claim 18, wherein the CD8+ T lymphocytes are co-cultured with allogeneic endothelial cells in the presence of interleukin-2 prior to preparing the sample and the reference sample with endothelial target cells and the CD8+ T lymphocytes.

20. The method according to claim 18, wherein at least one further pharmacologically active component is added to the sample and/or the reference sample.