TREATMENT METHOD FOR KIDNEY TRANSPLANT REJECTION

Abstract

Disclosed is a method for treating or preventing kidney transplantation rejection in a patient by administering to the patient autologous, ex vivo-modified and expanded CD4.sup.+CD25.sup.+Foxp3.sup.+CD127.sup.low T reg cells.

Claims

1. A method of treating and preventing kidney transplantation rejection in a mammalian subject, comprising administration to the subject a pharmaceutical formulation comprising: a therapeutically effective amount of ex vivo-modified and expanded, autologous regulatory CD4+CD25+ Foxp+CD127low T cells (T reg); and a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the T reg cells in the pharmaceutical formula have been derived from the peripheral blood of the subject to be treated.

3. The method of claim 1, wherein T reg cells in the pharmaceutical formulation have been stimulated with a CD3 mAb, a CD28 mAb, TGF-1, and IL-2.

4. The method of claim 1, wherein the T reg cells are present at 110.sup.6 to 1.110.sup.7 reg cell/kg body weight of the subject to be treated.

5. The method of claim 4, wherein the T reg cells are present at 210.sup.6 to 810.sup.6 T reg cells/kg body weight of the subject to be treated.

6. The method of claim 4, wherein the T reg cells are present at 410.sup.6 to 710.sup.6 T reg cells/kg body weight of the subject to be treated.

7. The method of claim 4, wherein the T reg cells are present at 510.sup.6 to 710.sup.6 T reg cells/kg body weight of the subject to be treated.

8. The method of claim 1, wherein the carrier is an intravenous glucose/dextrose solution, Ringer's lactate solution, sodium lactate solution, or Rheopolyglukin.

9. The method of claim 1, further comprising administering a different kidney transplantation rejection therapeutic compound.

Description

DESCRIPTION OF THE FIGURES

[0020] The foregoing and other objects of the present disclosure, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0021] FIG. 1A is a scatter plot showing characteristic T reg markers in the population of donor mononuclear cells which are CD25.sup.+;

[0022] FIG. 1B is a scatter plot showing characteristic T reg markers in the population of donor mononuclear cells which are Foxp3;

[0023] FIG. 1C is a scatter plot showing characteristic T reg markers in the population of donor mononuclear cells which are CD27.sup.low;

[0024] FIG. 2A is a scatter plot showing characteristic T reg markers in the population of donor CD4.sup.+ T cells which are CD25.sup.+;

[0025] FIG. 2B is a scatter plot showing characteristic T reg markers in the population of donor CD4.sup.+ T cells which are Foxp3;

[0026] FIG. 2C is a scatter plot showing characteristic T reg markers in the population of donor CD4.sup.+ T cells which are CD27.sup.low;

[0027] FIG. 3A is a scatter plot showing the expression of CD25.sup.hi on CD4.sup.+ T cells after 6 days of cultivation;

[0028] FIG. 3B is a scatter plot showing the expression of Foxp3.sup.+ on CD4.sup.+ T cells after 6 days of cultivation;

[0029] FIG. 3C is a scatter plot showing the expression of CD27.sup.low on CD4.sup.+ T cells after 6 days of cultivation; and

[0030] FIG. 4 is a graphic representation showing the increase in total number of cells (gray columns) and the increase in the number of T reg cells (shaded columns) at different stages of cultivation.

DETAILED DESCRIPTION

[0031] Throughout this application, various patents, patent applications, and publications are referenced. The disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.

[0032] It is discovered that the number of T reg cells in a patient at risk for, or suffering from, kidney transplantation rejection is variable, and that there is an inverse relationship between the number of T reg cells in the peripheral blood of such patients and the risk of rejecting the kidney transplant. This can be determined by studying both the rejection process and the number of T reg cells in a group of at risk patients relative to the number of T reg cells in those patients which are not at risk. Thus, a reduced level and functional activity of T reg cells are now understood to play an important role in the initiation and progression of the rejection. On this basis, a method for treatment of kidney transplantation rejection was developed which includes the immune correction therapy comprising T reg cells. In addition, it has been determined that treatment with autologous, modified and expanded T reg cells can inhibit the activity of autoimmune, autologous, cytotoxic T and B cells in a patient suffering at risk of, or from, kidney transplantation rejection.

Definitions

[0033] For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

[0034] The articles a and an are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, an element means one element or more than one element.

[0035] The term or is used herein to mean, and is used interchangeably with, the term and/or, unless context clearly indicates otherwise.

[0036] The term about is used herein to mean approximately, in the region of, roughly, or around. When the term about is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term about or approximately is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0037] As used herein, the term T reg cells refers to regulatory T cells with markers CD4.sup.+CD25.sup.+Foxp3.sup.+CD27.sup.low.

[0038] As used herein, the term treating refers to reducing or alleviating risk of, and/or symptoms of, kidney transplantation rejection, and/or preventing the occurrence and/or the progression of kidney transplantation rejection.

[0039] The term preventing refers to inhibiting or stopping the rejection.

[0040] The term native refers to cells from the body that have not been cultured, modified, expanded, or treated with any compound other than a life-sustaining medium.

[0041] Ex vivo-modified and expanded refers to native cells removed from the body, contacted with certain factors and compounds for modification, and cultivated to proliferate. In the methods of the present disclosure, when cells treated in this way are returned to the body of the patient from which they were originally removed, they are referred to as autologous, ex vivo-modified and expanded cells.

[0042] As used herein, the term healthy donor refers to a mammal, such a human, of the same specie as the patient and who has not had a kidney transplant, is not at risk of rejecting a kidney transplant, does not have any blood-related and/or inflammatory disorder, and is considered by a physician to be in good health. The number of T reg cells in the blood of a healthy donor is used herein to determine the number of T reg cells that are administered to the patient.

2. Preparation of Autologous, Ex Vivo-Modified and Expanded T Reg Cells

[0043] Autologous, modified and expanded T reg cells administered to a patient at risk or just experiencing initial phase of kidney transplantation rejection according to the method of the disclosure are derived from the peripheral blood of the patient. A sample of blood is removed and the fractionated to obtain a mononuclear cell (MNC) fraction from which the CD4.sup.+ T cells are later isolated. The MNC fraction can be obtained from blood by any known method of blood fractionation. For example, density gradient centrifugation, e.g. Ficoll-Hypaque density gradient centrifugation, can be used which takes advantage of the density differences between MNC's and other elements found in the blood sample. MNC's and platelets collect on top of the Ficoll-Hypaque layer because they have a lower density than red blood cells and granulocytes which collect at the bottom of the Ficoll-Hypaque layer.

[0044] To obtain T cells with a regulatory function, cells in this mononuclear fraction can be exposed to antibodies specific for CD4, as such T cells test positive for this marker. CD4.sup.+ cells can then be separated from the MNC fraction, for example, using CD4 MicroBead columns (Myltenyi Biotec, Germany) exposed to a magnetic field. These CD4.sup.+ cells are then screened for various other surface markers (CD25, Foxp3, and CD127.sup.low) which are characteristic of T reg cells, for example, by antibody staining, as described above and in Example 1B below.

[0045] The selected CD4.sup.+ T cells are then cultivated and expanded ex vivo. For example, cultivation can be done by growing cells in a growth medium adapted for T cells (e.g., RMP1-1640) after the cells have been stimulated to proliferate (e.g., by exposure to allogenic, antigen producing cells treated with mitomycin C, or TGF-Bl and IL-2).

[0046] To determine if the autolohous ex vivo-modified and expanded T reg cells have comparable suppressor activity relative to the native cells (circulating in blood), these cells are tested for their ability to suppress the proliferation of certain target cells involved in the autoimmune process. This can be done using any assay involving contacting certain selected target cells (e.g., those in a mixed lymphocyte sample) with the expanded T reg cells, and measuring the target cell's ability to proliferate.

3. Pharmaceutical Formulation

[0047] To prepare the pharmaceutical composition, the autologous ex vivo-modified and expanded T reg cells having suppressor activity are suspended in a pharmaceutically acceptable carrier. This can be accomplished, e.g. by washing them twice in PBS, centrifuging them, and suspending the cell pellet in the carrier.

[0048] The phrase pharmaceutically acceptable carrier is employed herein to refer to liquid solutions which are, within the scope of sound medical judgment, suitable for use in contact with the live T reg cells without affecting their activity, and without being toxic to the tissues of human beings and animals or causing irritation, allergic response, or other complications, commensurate with a reasonable benefit/risk ratio. A useful pharmaceutically acceptable carrier may be an injectable solution which is biocompatible with the T reg cells and does not reduce their activity or cause their death.

[0049] Non-limiting examples of materials which can serve as pharmaceutically acceptable carriers include a solvent or dispersion medium containing, for example, sterile intravenous glucose/dextrose sugar solutions, Ringer's lactate or compound sodium lactate solution.

[0050] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example Clindamycin, Fluconazole, and/or Amphotericin B. Sterile injectable solutions of expanded T reg cells can be prepared by incorporating the live cells in the required amount in an appropriate carrier.

[0051] The number of autologous, modified and expanded T reg cells which can be combined with a carrier material to produce a single-dosage form will vary depending upon the subject being treated, the particular mode of administration, the degree of risk of developing transplant rejection, the degree of kidney transplantation rejection symptoms, and/or the number of modified and expanded T reg cells in the subject's peripherical blood, among others. Ultimately, the number of T reg cells in the pharmaceutical composition is that number that causes a therapeutic effect when administered to the patient. For example, the effective amount of the T reg cells may be about 110.sup.6 to about 1.110.sup.7 T reg cells/kg body weight, about 210.sup.6 to about 810.sup.6 T reg cells/kg body weight, about 410.sup.6 to about 710.sup.6 T reg cells/kg body weight, or about 510.sup.6 to about 710.sup.6 T reg cells/kg body weight.

4. Therapeutic Administration

[0052] Administration of the formulation containing the autologous T reg cells is useful to prevent or treat and/or to inhibit the activity of autoimmune, autologous, cytotoxic T and B cells in a patient at risk for, or suffering from, kidney transplantation rejection. This step comprises administering a therapeutically effective amount of autologous, modified and expanded T reg cells to the patient.

[0053] Methods of administration of these T reg cells in the pharmaceutical composition according to the disclosure described herein can be by any of a number of methods well known in the art. These methods include systemic or local administration by injection. Exemplary routes of administration include intravenous, intramuscular, intraperitoneal, or subcutaneous injection, and any combinations thereof.

[0054] The initial administering step may be a single administration 1 to 2 weeks after transplant or may comprise multiple administrations every 1, or 2 to 4 weeks after the initial administration. The initial administrating steps may be performed every 1 to 8 weeks. The number of initial administering steps at the start of treatment depends on the initial level of T reg cells in a patient's peripheral blood. The goal is to increase T reg cell number in the patient's peripheral blood until it is at the level of a healthy donor. This is determined by measuring the number of T reg cells in the peripheral blood of the patient before and after administering the expanded and modified T reg cells, and comparing that number to the number of T reg cells in the peripheral blood of a healthy patient. At the start of therapy, 1 to 8, 2 to 7, 3 to 6, 4 to 6, or 3 to 5 T reg cell injections can be administered every 1, 2 to 4, or 3 to 4 weeks.

[0055] In addition, in some cases, an additional administering step may be performed every 3 to 6 months after the start of treatment, or at the end of the administration of the initial multiple treatments. However, a physician may determine that administration of the autologous T reg cells may be daily, weekly, or monthly. Even a single bolus provided about 1 or 2 weeks after transplant can significantly improve the patient's condition.

[0056] In order to determine the number of T reg cells in a patient's blood, a sample is taken for measurement. Any method that enables the measurement of the number of T reg cells can be used. For example, the flow cytometry analysis can be performed. Measurement of the number of T reg cells can be done before and after each initial and secondary administering step(s), and further, can be done months after the initial; and any secondary administering step(s), for example, every two months. The T reg cell-containing pharmaceutical composition can also be administered as part of a combination therapy with other agents to prevent or treat kidney transplantion rejection.

[0057] Combination therapy refers to any form of administration combining two or more different therapeutic compounds, where the second compound is administered while the previously administered autologous, modified and expanded T reg cells are still effective in the body (e.g., the two therapeutics are simultaneously effective in the patient, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered in separate formulations, either simultaneously or sequentially. Thus, a patient who receives such treatment can have a combined (conjoint) effect of different therapeutic compounds.

[0058] The following examples provide specific exemplary methods of the invention, and are not to be construed as limiting the invention to their content.

EXAMPLES

Example 1

[0059] Isolation and Ex Vivo-Modification and Expansion of T Reg Cells

[0060] All the manipulations are performed under aseptic conditions in a Laminar Flow Class II Biosafety Cabinet which is located in a sterile clean room following to GMP regulations.

A. Blood Drawing

[0061] Peripheral blood (40 ml-50 ml) was taken from the ulnar vein of patients and placed into sterile tubes (Vacutainer, BD, USA). 20 ml-30 ml blood (vacutainer glass serum tubes) was kept at room temperature (RT) for 2 hours, and then centrifuged at 350 g for 15 min. The supernatant was collected into sterile tubes (Falcon, 15 ml conical tubes), which were incubated for 40 min at 56 C. to inactivate complement. The serum was bottled in 1.5 ml vials (Coming, USA) and frozen at 20 C.

B. Isolation of MNC's

[0062] The blood was transferred from tubes with the anticoagulant into 50 ml tubes, dilute 1:1 with Phosphate-buffered Saline (PBS, Ca.sup.+2 Mg.sup.+2 free, Gibco, United Kingdom). In order to separate lymphocytes, 35 ml MNC suspension was layered over 15 ml of a gradient solution (LimphoSep, d=1.077 g/ml, MP Biomedicals, USA) in 50 ml conical tubes (Falcon, USA). The tubes were centrifuged at 400 g for 30 min at 20 C. The upper layer was aspirated off, leaving the MNC layer, which was transferred to new 50 ml conical tubes. The tubes were filled with buffer and centrifuged at 300 g for 10 min. The cell pellet was resuspended in 50 ml PBS, combined in one tube, and then centrifuged at 300 g, 20 C. for 10 min. This procedure was repeated, and the cell pellet was resuspended in an appropriate amount of buffer.

[0063] For an estimation of initial CD4.sup.+CD25.sup.+Foxp3.sup.+CD27.sup.low T reg cell numbers, the MNC population was stained with anti-CD4.sup.+, anti-CD25.sup.+, anti-Foxp3.sup.+, and anti-CD127.sup.+ mAbs (Miltenyi Biotec, Germany; eBioscience, USA). The cells were detected by flow cytometry using a MACsQuant (Miltenyi Biotec, Germany).

[0064] FIGS. 1A-1C show a representative example of characteristic T reg cell markers in the donor's MNCs: 5.7% of CD4.sup.+ T cells co-expressed CD25.sup.+ (FIG. 1A); 3.4% of CD4.sup.+ T cells co expressed Foxp3 (FIG. 1B); and 3.8% of CD4.sup.+ T cells co-expressed CD127.sup.low (FIG. 1C).

C. Isolation of CD4.SUP.+ T Cells

[0065] In order to isolate CD4.sup.+ T cells, MNC were magnetically labeled with CD4+ mAbs according to the MACS Miltenyi Biotec (Germany) procedure. The immune phenotype of isolated CD4.sup.+ T cells was estimated by flow cytometry. In average, 944% (n=19) of the isolated cells were CD4.sup.+ T cells.

[0066] Expression of T cell markers on these cells is shown in FIGS. 2A-2C from one representative experiment (total 19): 97.5% of cells expressed CD4.sup.+, and 12.6% of these CD4.sup.+ cells co-expressed CD25.sup.+ (FIG. 2A); 6.3% of these CD4.sup.+ cells co-expressed Foxp3.sup.+ (FIG. 2B); and 7.2% of these CD4.sup.+ cells co-expressed CD127.sup.low (FIG. 2C).

D. Modification and Expansion of T Reg Cells

[0067] The medium used for the T reg cell culture was RPMI-1640 which contains phenol red, L-glutamine, and 25 MM HEPES (Gibco, UK) with the addition of both 5-10% autologous serum and 1% pen/strep (Gibco, UK). This medium was supplemented with 1 ng/ml-50 ng/ml transforming growth factor 1 (TGF 1) (R&D Systekidney transplantion rejection, UK), 10 U/ml-1000 U/ml interleukin-2, (IL-2, R&D Systekidney transplantion rejection, UK), 0.1 g/ml-10 g/ml mouse anti-human CD3 mAbs (Med biospecter, RF), and 0.1 g/ml-10 g/ml mouse anti-human CD28 mAbs (BD Pharmingen, USA). Expanded CD4+ T cells were cultured at 37 C. in 5% CO.sub.2 for 6 to 8 days in flasks (either 25 cm.sup.2 or 75 cm.sup.2) with all supplements. After 3 to 4 days, IL-2, TGFB1, anti-human CD3mAbs, and anti-human CD28 mAbs were added.

E. Phenotypic Characterizations of T Reg Cells after Modification and Expansion Ex Vivo

[0068] Modified and expanded cells were characterized at the end of culture. Flow cytometry was used to estimate the total numbers of live cells and the proportion of CD4.sup.+CD25.sup.+Foxp3.sup.+CD27.sup.low cells in the cell suspension. To assure that the endotoxin levels in cell preparations were negligible, aliquots were tested with the Limulus assay kit (Sigma-Aldrich, USA), according to the manufacturer's protocols.

[0069] Table 1 shows the results of flow cytometry of initial CD4.sup.+ T cells and the same cells after 6 to 7 days of culture with modifying/stimulating molecules.

TABLE-US-00001 TABLE 1 Marker Measurement in Marker Measurement Modified and Markers in Initial cells Expanded cells CD4.sup.+ 93.9 4.5 99.8 0.2 CD4.sup.+CD25.sup.hi 15.7 4.0 95.9 2.4 CD4.sup.+CD25.sup.+CD62L.sup.+ 18.8 9.0 54.6 3.8 CD4.sup.+CD25.sup.+Foxp3.sup.+ 6.1 4.8 89.6 3.2 CD4.sup.+CD25.sup.+CD152.sup.+ 5.4 2.7 93.8 3.0 CD4.sup.+CD25.sup.+CD127.sup.low 6.7 4.1 91.3 3 .2

[0070] FIGS. 3A-3C show a representative sample of T reg cells expression after 6 days of ex vivo culture. 99.6% CD4.sup.+ T cells co-expressed CD25.sup.hi (FIG. 3A); 91.7% CD4.sup.+ T cells co-expressed Foxp3 (FIG. 3B); and 92.3% CD4.sup.+ cells co-expressed CD127.sup.low (FIG. 3C).

[0071] During the 6 days of propagating CD4.sup.+ T cells (obtained from 19 donors), the total amount of cells increased 27.27 0.3 X, whereas the number of T reg cells CD4.sup.+CD25.sup.+Foxp3.sup.+ increased 1272470 X (FIG. 4).

E. Functional Capacity of Modified and Expanded T Reg Cells

[0072] To determine if ex vivo-modified and expanded T reg cells keep their own suppressive ability, their suppressive capacity to inhibit proliferation of target cells in a mixed lymphocyte reaction (MLR) was compared initially and after the expansion of T reg cells.

[0073] To this end, autologous target cells (CD4.sup.+, CD4.sup.+CD25.sup.+) were isolated using the magnetic beads selection method (Miltenyi Biotec), stained with carboxyfluorescein succinimidyl ester (CFSE, Fluka, USA), and cultivated with or without equal numbers (1:1) of native T reg cells isolated from human blood or induced, ex vivo-modified and expanded T reg cells. Either T cells CD4.sup.+CD2S.sup. T cells or CD4.sup.+ T cells were stimulated by 5 g/ml (CD3 mAbs and allogeneic MNC treated with mitomycin C and depleted of CD3+ T cells by the magnetic bead selection method (Miltenyi Biotec).

[0074] After 4 to 5 days of culture, cell proliferation was estimated by measurement of a reduction of 5(6) Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) in proliferating cells.

[0075] The functional activity of T reg cells isolated from the peripheral blood of kidney transplantation rejection patients is found to be substantially reduced.

[0076] Thus, the number of T reg cells in a patient at risk for, or suffering from, kidney transplantation rejection is variable. This can be determined by studying the immuno-phenotype of these cells within one group of patients in both the relapse stage.

Example 2

Treatment of Patients Experiencing Kidney Transplantation Rejection Ex Vivo

[0077] Five patients at risk or suffering from the initial stage of kidney transplantation rejection are treated with autologous, ex vivo-modified and expanded CD4.sup.+CD25.sup.+CD127.sup.lowT reg cells.

[0078] Patients undergoing treatment do not receive either steroids or immunotherapy for at least 3 consecutive months. All the patients signed Consent Agreement before taking part in the study.

[0079] Increased numbers of circulating T reg cells and decrease symptoms of kidney rejection are shown in patients 4 days after injection of autologous, ex vivo-modified and expanded CD4.sup.+CD25.sup.+Foxp3.sup.+ T reg cells.

EQUIVALENTS

[0080] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.