RU2454247C1 - Method for prevention of acute graft-versus-host disease following marrow allografting - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, more specifically to haematology, and is applicable for immune response modulation, particularly for prevention of acute graft-versus-host disease following marrow allografting. Donor multipotent mesenchymal stromal cells recovered from bone marrow, cultured on a human blood plasma medium enriched in platelet lysate are introduced intravenously in the amount of 1x10⁶ grafted cells per one kg of patient's body weight at the moment of leukocyte recovery in patient's peripheral blood following marrow allografting min. 1.0×10⁹/l.

EFFECT: method provides effective prevention of acute graft-versus-host disease following marrow allografting.

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Description

The invention relates to medicine, namely to hematology, and can be used to modulate the immune response, in particular for the prevention of acute transplant versus host (GVHD) reaction after allogeneic bone marrow transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is used to treat many oncohematological diseases, as well as aplastic anemia and solid tumors, and is often the only method to completely cure patients. The graft versus host (GVHD) reaction is the main complication that occurs after transplantation of allogeneic hematopoietic stem cells associated with damage to recipient organs and tissues by immunocompetent T-lymphocytes of the donor. The aim of many works is to achieve successful engraftment of donor hematopoietic cells, accompanied by an antitumor effect and at the same time not leading to GVHD. Despite all efforts, in 10-80% of patients after transplantation of allogeneic hematopoietic stem cells, acute GVHD develops during the first 100 days after HSCT. The main target organs in this complication are the skin, liver and gastrointestinal tract. The graft versus host reaction, especially its resistant form, remains the leading cause of death for recipients after HSCT. Approximately half of patients with treatment-resistant forms of GVHD die, usually due to infections associated with this process. Prevention of the graft versus host reaction using immunosuppressive therapy or removal of donor lymphocytes from transplanted hematopoietic cells is not always completely successful. Treatment of GVHD includes the use of immunosuppressive drugs such as glucocorticoids, cyclosporin / tacrolimus, antithymocyte globulin, monoclonal antibodies. However, all these agents increase the risk of transplant non-healing, the development of infectious complications and relapse of the original disease.

A chronic graft versus host (hRTP) reaction usually develops 100-500 days after HSCT in about 50% of patients and is accompanied by symptoms similar to autoimmune diseases. The incidence of hRTP is higher in patients previously undergoing acute GVHD. Modern therapy for the chronic transplant versus host reaction includes immunosuppressants and cytostatic drugs. As with GVHD, prolonged treatment with immunosuppressive drugs increases the risk of infections and relapses. In this regard, the use of new methods of treatment for GVHD is extremely important.

For human cells, it was proposed to define mesenchymal stem cells (MSCs) as fibroblast-like bone marrow cells that adhere to plastic and are able to differentiate in the bone, cartilage, and fat directions under the action of appropriate in vitro inducers (Caplan, (1991) J. Orthop.Res., 9 : 641-650). For cells that meet this definition, it is impossible to prove self-sustainability. In this regard, it was proposed to call such cells multipotent mesenchymal stromal cells (MMSCs) (Horwitz et al., (2005) Cytotherapy., 7: 393-395), and use the term MSCs only for cells that have proven self-sustainability. In cell therapy, mesenchymal progenitor cells are used, which are essentially MMSCs.

MMSCs isolated from bone marrow have immunosuppressive properties, as demonstrated in in vivo and in vitro studies. MMSCs do not express hematopoietic cell markers such as CD45, CD34, CD3, CD14, whereas CD29, CD90, CD73, CD105, CD106, CD146 and CD273 are markers of MMSCs (Majumdar et al., (2003) J. Biomed. Sci. 10: 228-241). MMSCs are multipotent cells capable of differentiating into various mesenchymal differentiation lines, including adipocytes, chondrocytes, and osteocytes (Pittenger et al., (1999) Science, 284: 143-147). MMSCs secrete various cytokines, growth factors, and extracellular matrix molecules that play an important role in the proliferation and maturation of hematopoietic stem cells (Jones et al., (2008) Exp. Hematol., 36: 733-741). The study of the properties of MMSCs during cultivation in the in vitro system showed that these cells are not immunogenic and possess immunosuppressive properties. The ability to obtain a large number of MMSCs needed for therapeutic needs during cultivation led to a rapid transition from an experimental in vitro study to the use of these cells in Phase P clinical protocols (Jones et al., (2008) Exp. Hematol., 36: 733-741 )

MMSCs are obtained from various embryonic and adult tissues, which include bone marrow, adipose tissue, dermis, cord blood cells, placenta, and amniotic fluid. The main way to obtain them is to isolate a population of fibroblast-like cells adhering to the plastic of culture dishes (patent numbers RU 2331670 C1, 08.20.2008, RU 2323252 C1, 04.27.2008, RU 2303632 C1, 07.27.2007, RU 2280462 C2, 07.27.2006, RU 2252252 C1, 05.20.2005, WO 1999047163, 09/23/1999, WO 2005093044, 10/06/2005, WO 2007143139, 12/13/2007, WO 2009040666, 04/02/2009).

MMSCs have been shown to avoid allogeneic rejection in humans (Aggarwal et al., (2005) Blood, 105: 1815-1822), (Le Blanc et al., (2004) Scand.J. Immmunol., 60: 307-315 ) and can be transplanted without special immunosuppression (Majumdar et al., (2003) J. Biomed.Sci., 10: 228-241).

The presence of immunomodulatory properties in MMSCs allows them to be used for the treatment of autoimmune diseases (RU 2298410 C1, 05/10/2007, WO 2005093044, 10/06/2005, WO 2008042174, 04/10/2008). Some studies used a biograft for the treatment of autoimmune and rheumatic diseases containing mesenchymal stem cells (MSCs) obtained from fetal, donor or autologous material, the tissue was disaggregated, the resulting cell suspension was resuspended and cultured on a growth medium containing transferrin, insulin, factor growth of fibroblasts and heparin before the accumulation of mature stroma cells in the culture. A method for the treatment of autoimmune and rheumatic diseases consisted in the intravenous drip of MSCs from 50 to 500 million in 50-100 ml of physiological saline or infucol (RU 2298410 C1, 05/10/2007). In other cases, MSCs were cultured on growth medium containing 10% fetal calf serum until the required number of cells was accumulated in the culture. The treatment method consisted of intravenous, intraperitoneal, or local (directly to the organ) administration from 10 thousand to 100 million MSCs (WO 2005093044, 10/06/2005, WO 2008042174, 04/10/2008).

Recently, important data have been obtained on the study of the immunomodulating properties of MMSCs, with the help of which these cells can prevent or treat GVHD.

Based on the data obtained, MMSCs were started to be used in the clinic (Le Blanc et al., (2004) Lancet, 363: 1439-1441), a large number of patients were treated with MMSCs and encouraging results were obtained (Jorgensen C. et al., (2003 ) Gene Therapy, 10, 928-931). Le Blanck et al. (Ringden et al., (2006) Transplantation, 81: 1390-1397) have shown that the administration of MMSCs is associated with a significant improvement in the condition of patients without significant side effects. However, the mechanism of action of MMSC, leading to a clinical improvement in the condition of patients, remains unknown, and work on animal models has not yet yielded unambiguous results. Various animal studies have found that MMSCs highly effective in preventing the development of GVHD, however, are not the preferred treatment for the treatment of severe GVHD already developed (Tisato et al., (2007) Leukemia, 21: 1992-1999) (Sudres et al. , (2006) J. Immunol., 176: 7761-7767). Toubai T. et al., Mesenchymal Stem Cells for Treatment and Prevention of Graft-Versus-Host Disease After Allogeneic Hematopoietic Cell Transplantation, Current Stem Cell Research & Therapy, 2009, 4, 252-259, which lists 6 prevention works The acute transplant versus host reaction in mouse models, in particular the work (Tisato et al., (2007) Leukemia, 21: 1992-1999), is the closest analogue (prototype) of the claimed method. In this analogue, the signs that coincide with the essential features of the claimed invention are (1) the use of MMSC for (2) the prevention of the development of GVHD. However, the work was performed on MMSCs of mice or MMSCs obtained from human umbilical cord blood; immunodeficient mice were used as recipients, which impedes the achievement of the technical result provided by the present invention. Currently, all published work on the prevention of acute transplant versus host reaction has been performed in mice. There is no work on the prevention of an acute transplant versus host reaction with mesenchymal cells in humans.

For the treatment and prophylaxis of GVHD in foreign patents, it is proposed to use both MMSCs themselves and an air-conditioned environment in which MMSCs were pre-cultured, as well as lysates of these cells, as immunosuppressive agents (WO 2001032189, 05/10/2001, WO 1999047163, 09/23/1999, WO 2009134429, 11/05/2009). In some cases, the use of MMSCs was combined with standard immunosuppressive therapy (WO 2007089627, 08/09/2007, WO 2007143139, 12/13/2007).

All the described methods for the preparation of MMSCs used either a serum-free medium or fetal calf serum (ETS). It is known that on a serum-free medium, cells grow worse and age faster. The use of ETS is associated, firstly, with the contact of human cells with a product of animal origin, which can lead to various complications (Samuelsson et al., (2009) Cytotherapy., 11: 129-136), and secondly, the remnants of ETS that enter the body of patients, they develop an immune response. Thus, in the treatment of osteogenesis imperfecta in a child who received MMSC from his father several times, grown in an environment with ETS, antibodies to ETS were developed that neutralized the effect of the graft (Horwitz et al., (1999) Nat.Med., 5: 309-313 )

These complications can be avoided by using platelet-rich plasma instead of ETS. Lange et al. (2007) J. Cell Physiol, 213: 18-26) showed that the addition of 5% platelet lysate obtained from platelet concentrate to the culture medium provides MMSC growth with an efficiency similar to that in the presence of ETS.

The technical result of the claimed invention based on the developed cell technology is to increase the effectiveness of the prophylaxis of GVHD by reducing complications when using MMSCs from donor bone marrow cells.

The task of preventing GVHD is achieved by using multipotent mesenchymal stromal donor cells (MSCSD) grown on a medium with human blood plasma enriched in lysed platelets. In this case, the cells are injected intravenously in an amount of 1 × 10 ⁶ per kg of patient weight at the time of recovery of leukocytes in the patient’s peripheral blood after transplantation of allogeneic bone marrow of more than 1.0 × 10 ⁹ / L.

The human blood plasma enriched with platelets (platelet concentration 0.5-1.9 × 10 ⁹ / ml) is obtained, preferably, by thawing a platelet concentrate pre-frozen at -70 ° C, followed by precipitation of platelet lysate at 12,000 rpm for 15 minutes.

MSCDs can be obtained from nucleated donor bone marrow cells.

Nucleating donor bone marrow cells are obtained by resuspending the bone marrow in a culture medium with 0.1% methylcellulose, incubating the mixture at room temperature for 40 minutes and then centrifuging the erythrocyte-free fraction at 1500 rpm for 10 minutes.

Nucleated cells are cultured in αMEM medium with 4% platelet-rich plasma, 200 mM L-glutamine and antibiotics at a concentration of 120,000 cells per cm ^{2 of} the bottom area of the vial.

Multipotent donor mesenchymal stromal cells that have reached confluence are removed from the substrate using 0.025% trypsin and passaged at a concentration of 4500 cells per 1 cm ^{2 of the} bottom of the vial.

After removal from the substrate, the MMSCD can be centrifuged at 1500 rpm for 10 minutes and resuspended in polyglucin in the required amount.

MSCD is administered in an amount of 1 × 10 ⁶ / kg of patient weight intravenously for 10 minutes.

MSCDs can be used both immediately after their preparation and after thawing of cells previously frozen in liquid nitrogen vapors in an environment with 10% dimethyl sulfoxide.

Multipotent donor mesenchymal stromal cells can be introduced for the prevention of GVHD during the recovery of hematopoiesis after transplantation of stem hematopoietic cells (HSCT), determined by restoring the number of leukocytes in its peripheral blood to more than 1.0 × 10 ⁹ / L. The achieved result is to obtain MMSCSD compatible with the patient’s immune system under optimal cultivation conditions, as well as to administer them to the patient in the required quantity and at a strictly defined time.

Example 1

Patient B., 20 years old, diagnosed with chronic myelogenous leukemia, weight 56 kg. Allogeneic bone marrow transplantation was performed on 12/17/08. After 27 days in the patient’s peripheral blood, leukocyte recovery to 1.4 × 10 ⁹ / L was noted. MSCSD in a dose of 70 × 10 ⁶ (1.25 × 10 ⁶ / kg) was introduced on January 14, 2009. No complications were noted. There were no signs of a graft versus host reaction. The observation period is 1 year 9 months.

Example 2

Patient K., 22 years old, diagnosed with acute lymphoblastic leukemia, weight 48 kg. Allogeneic bone marrow transplantation was performed on 02.10.09. After 20 days in the patient’s peripheral blood, leukocyte recovery to 1.6 × 10 ⁹ / L was noted. MSCSD was transfused on March 11, 2009, with a dose of 53 × 10 ⁶ (1.1 × 10 ⁶ / kg). An increase in temperature to 37.8 ° C was noted. There were no signs of a graft versus host reaction. The observation period is 1 year 8 months.

Example 3

Patient P., 29 years old, diagnosed with acute myeloid leukemia, weight 60 kg. Allogeneic bone marrow transplantation was performed on 02.20.09. After 24 days in the patient’s peripheral blood, leukocyte recovery to 1.0 × 10 ⁹ / L was noted. March 23, 2009 introduced MSCSD in a dose of 60 × 10 ⁶ (1.0 × 10 ⁶ / kg). There was an increase in temperature to 38.1 ° C with chills. There are no signs of GVHD. The observation period is 1 year 8 months.

Example 4

Patient R., 46 years old, with a diagnosis of acute myeloid leukemia, weight 56 kg. Allogeneic bone marrow transplantation was performed on 03/27/09. After 20 days in the patient’s peripheral blood, leukocyte recovery to 1.0 × 10 ⁹ / L was noted. 05/20/2009 MMSKD was introduced at a dose of 60 × 10 ⁶ (1.07 × 10 ⁶ / kg). There was an increase in temperature to 38.8 ° C with chills. There are no signs of GVHD. The observation period is 1 year 7 months.

Example 5

Patient R., 47 years old, diagnosed with myelodysplastic syndrome, weight 70 kg. Allogeneic bone marrow transplantation was performed on 12/08/09. After 23 days in the patient’s peripheral blood, leukocyte recovery to 1.2 × 10 ⁹ / L was noted. 01/11/2010 introduced MSCSD in a dose of 65 × 10 ⁶ (0.93 × 10 ⁶ / kg). There was an increase in temperature to 37.8 ° C with chills. There are no signs of GVHD. The observation period is 10 months.

In total, 13 patients were administered MMSCSD for the prevention of GVHD. All 13 did not show the development of GVHD - II or more, in two patients, prior to the management of MMSC, there were signs of an acute graft versus host I st reaction that completely regressed after administration of multipotent mesenchymal stromal donor cells. Of the 12 patients who were not injected with MMSCD during the period of recovery of the number of leukocytes in the peripheral blood of more than 1.0 × 10 ⁹ / L, 9 developed advanced GVHD II and more. There was no difference in infectious complications, the number of relapses of the underlying disease, cases of transplant rejection and general mortality in the groups of patients who underwent and did not carry out the prophylaxis of GVHD with the help of MMSC.

Thus, timely administration of MMSC is an effective prevention of the development of GVHD in patients after hematopoietic stem cell transplantation.

Claims (1)

A method for the prevention of an acute transplant versus host reaction during allogeneic bone marrow transplantation, comprising administering to a patient multipotent mesenchymal stromal donor cells obtained from donor bone marrow, characterized in that multipotent mesenchymal stromal donor stromal cells obtained from donor bone marrow grown on plasma medium are used human blood enriched lysed platelets, which is administered intravenously in an amount of 1 × ¹⁰ June transplantable cells per 1 kg of patient weight th at the time of restoration of leukocytes in the peripheral blood of a patient following transplantation of allogeneic bone marrow more 1,0 × 10 ⁹ / L.