Patent Application
20200147138
The invention relates to medicine, immunology, and more specifically to the prevention and/or treatment of kidney transplant rejection.
Kidney transplantation is indicated at end-stage renal disease (ESRD). This is defined as a glomerular filtration rate less than 15 ml/min/1.73 m2. Common diseases leading to ESRD include malignant hypertension, infections, diabetes mellitus, and focal segmental glomerulosclerosis. Genetic causes include polycystic kidney disease, a number of inborn errors of metabolism, and some autoimmune conditions.
Diabetes is the most common known cause of needing kidney transplantation, accounting for approximately 25% of those in the US. The majority of renal transplant recipients are on dialysis at the time of transplantation. However, individuals with chronic kidney disease who have a living donor available may undergo pre-emptive transplantation before dialysis is needed.
The newly transplanted kidney usually begins functioning immediately. Living donor kidneys normally require three to five days to reach normal functioning levels, while cadaveric donations stretch that interval to seven to fifteen days.
Transplant rejection occurs when the donor kidney is rejected by the recipient's immune system, which destroys the transplanted tissue. Transplant rejection can be decreased by using a kidney from a donor with antigenic similarities with the recipient.
The chance of rejection can also be reduced by the use of immunosuppressant drugs after transplant. Immunosuppressant drugs are used to suppress the immune system from rejecting the donor kidney. However, these drugs must be taken for the rest of the recipient's life. A common medication regimen today is a mixture of tacrolimus, mycophenolate, and prednisolone. Alternatively, some recipients may instead take ciclosporin, sirolimus, or azathioprine.
The risk of early rejection of the transplanted kidney is increased if corticosteroids are avoided or withdrawn after the transplantation. Ciclosporin and tacrolimus cause nephrotoxicity and can result in iatrogenic damage to the newly transplanted kidney. Thus, blood levels of both must be monitored closely and if the recipient seems to have declining renal function or proteinuria, a biopsy may be necessary to determine whether this is due to rejection or to ciclosporin or tacrolimus intoxication.
Antibody treatments such as monoclonal anti-IL-2Rα receptor antibodies, polyclonal anti-T cell antibodies, and monoclonal anti-CD20 antibodies have been used. However, cases refractory to immunosuppressive or antibody therapy have occurred and may have to be treated with photopheresis, or extracorporeal photoimmune therapy (ECP), to remove antibody molecules specific to the transplanted tissue.
In addition, treatment with a bone marrow transplant, which replace the transplant recipient's immune system with the donor's, allows the recipient accepts the new organ without rejection. However, there is risk of graft-versus-host disease if T cells within the recipient's marrow recognize the new host tissues as foreign and destroy them.
Thus, there is a need for improved methods of treatments which reduce kidney transplantation rejection.
It is being discovered that there is an inverse relationship between the number of CD4+CD25+Foxp3+ (T reg) cells in the peripheral blood of kidney transplant patients who are at risk for transplant rejection or that are experiencing kidney transplantation rejection and the degree of severity of the condition. This discovery has been exploited to develop the present method of preventing and/or treating kidney transplantation rejections. Administering autologous, ex vivo-modified and expanded CD27low regulatory T reg cells starting about a week after transplant lowers the level of autoimmune activity of certain T cells, thereby reducing symptoms of rejection and maintaining a non-rejection phase indefinitely or for at least longer periods of time than what is seen on average without treatment.
In one aspect, the disclosure provides a method of preventing or treating kidney transplantation rejection in a patient in need thereof, comprising administering a therapeutically effective amount of autologous, modified and expanded T reg cells to the patient about one or two weeks after transplant.
In some embodiments, the administering step comprises administering the therapeutically effective amount of these T reg cells more than one time at the start of treatment to increase the number of T reg cells in patient's peripherical blood to a number comparable to number in the peripherical blood of healthy donors. In some embodiments, the administering step comprises administering a therapeutically effect amount of autologous, modified and expanded T reg cells about 1 to 7 times at the start of treatment.
In some embodiments the method further comprising measuring the number of T reg cells in the peripherical blood of the patient before and after each administering step. In particular embodiments, the number of T reg cells in a patient's peripherical blood is measured weekly, monthly, 1 to 3 months, 2 to 3 months, or every week, after the initial administering step.
In particular embodiments, the method further comprises a second administering step if the number of T reg cells measured in the blood of a patient after the first administering step is less than the number of T reg levels in blood of a healthy donor.
In certain embodiments, the number of autologous T reg cells administered at one time is about 1×106 to about 1.1×107 per kg body weight to the patient. In many embodiments, the autologous T reg cells are administered by subcutaneous, intravenous, and/or intramuscular injection.
Also provided by the present disclosure is a method of inhibiting the activity of autoimmune, autologous, cytotoxic T and B cells in a patient at risk for, suffering from kidney, or transplantation rejection, comprising: administering a therapeutically effective amount of autologous, modified and expanded T reg cells to the patient. In some embodiments, the administering step is performed more than one time throughout the life of the patient. In certain embodiments, the administering step is performed weekly, every 1 to 7, 2 to 6, 3 to 6, or 4 to 5 months after the first administering step.
In many embodiments, the administering step comprises administering about 1×106 to about 1.1×107 autologous T reg cells per kg body weight to the patient. In some embodiments, the autologous, modified and expanded T reg cells are administered by subcutaneous, intravenous, and/or intramuscular injection.
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:
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.
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.
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.
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.
The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
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%.
As used herein, the term “T reg cells” refers to regulatory T cells with markers CD4+CD25+Foxp3+CD27low.
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.
The term “preventing” refers to inhibiting or stopping the rejection.
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.
“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”.
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.
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+ 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.
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+ 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+ cells are then screened for various other surface markers (CD25, Foxp3, and CD127low) which are characteristic of T reg cells, for example, by antibody staining, as described above and in Example 1B below.
The selected CD4+ 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).
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.
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.
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.
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.
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.
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 1×106 to about 1.1×107 T reg cells/kg body weight, about 2×106 to about 8×106 T reg cells/kg body weight, about 4×106 to about 7×106 T reg cells/kg body weight, or about 5×106 to about 7×106 T reg cells/kg body weight.
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.
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.
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.
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.
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.
“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.
The following examples provide specific exemplary methods of the invention, and are not to be construed as limiting the invention to their content.
Isolation and Ex Vivo-Modification and Expansion of T Reg Cells
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.
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.
The blood was transferred from tubes with the anticoagulant into 50 ml tubes, dilute 1:1 with Phosphate-buffered Saline (PBS, Ca+2 Mg+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.
For an estimation of initial CD4+CD25+Foxp3+CD27low T reg cell numbers, the MNC population was stained with anti-CD4+, anti-CD25+, anti-Foxp3+, and anti-CD127+ mAbs (Miltenyi Biotec, Germany; eBioscience, USA). The cells were detected by flow cytometry using a MACsQuant (Miltenyi Biotec, Germany).
In order to isolate CD4+ T cells, MNC were magnetically labeled with CD4+ mAbs according to the MACS Miltenyi Biotec (Germany) procedure. The immune phenotype of isolated CD4+ T cells was estimated by flow cytometry. In average, 94±4% (n=19) of the isolated cells were CD4+ T cells.
Expression of T cell markers on these cells is shown in
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 μg/ml-10 μg/ml mouse anti-human CD3 mAbs (Med biospecter, RF), and 0.1 μg/ml-10 μg/ml mouse anti-human CD28 mAbs (BD Pharmingen, USA). Expanded CD4+ T cells were cultured at 37° C. in 5% CO2 for 6 to 8 days in flasks (either 25 cm2 or 75 cm2) 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
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+CD25+Foxp3+CD27low 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.
Table 1 shows the results of flow cytometry of initial CD4+ T cells and the same cells after 6 to 7 days of culture with modifying/stimulating molecules.
During the 6 days of propagating CD4+ T cells (obtained from 19 donors), the total amount of cells increased 27.2±7 0.3 X, whereas the number of T reg cells CD4+CD25+Foxp3+ increased 1272±470 X (
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.
To this end, autologous target cells (CD4+, CD4+CD25+) 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+CD2S− T cells or CD4+ T cells were stimulated by 5 μg/ml (CD3 mAbs and allogeneic MNC treated with mitomycin C and depleted of CD3+ T cells by the magnetic bead selection method (Miltenyi Biotec).
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.
The functional activity of T reg cells isolated from the peripheral blood of kidney transplantation rejection patients is found to be substantially reduced.
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.
Five patients at risk or suffering from the initial stage of kidney transplantation rejection are treated with autologous, ex vivo-modified and expanded CD4+CD25+CD127lowT reg cells.
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.
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+CD25+Foxp3+ T reg cells.
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.
This application claims priority to U.S. Provisional application No. 62/758,396, filed Nov. 9, 2018, the contents of which are hereby incorporated herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62758396 | Nov 2018 | US |
