KR101755260B1 - Immunosuppression Composition for allogeneic- and Xenogeneic Transplantation Comprising Regulatory T cell and CD40-CD40L blockade - Google Patents

Abstract

The present invention relates to a composition for suppressing an immune response comprising an immunoregulatory T cell (Regulatory T cell) and a CD40-CD40L binding inhibitor. In accordance with the present invention, the present invention allows pig islets transplanted into primates to survive for a long period of time and exhibits high reproducibility, so that it is possible to overcome various immune rejection reactions occurring in recipients of allogeneic or xenogeneous organs Can be effectively used.

Description

[0002] Immunosuppression Composition for allogeneic and Xenogeneic Transplantation Comprising Regulatory T cells and CD40-CD40L blockade [0156]

The present invention relates to a composition for suppressing an immune response comprising an immunoregulatory T cell and a CD40-CD40L binding inhibitor.

Islet is a cell mass consisting of alpha, beta, and delta cells. It is a name attached to the pancreas as if it were an island. Delta cells are important cells that regulate blood sugar in the blood by secreting insulin. In order to treat type 1 diabetes, allogeneic liver transplantation methods are currently underway in which only the insulin-secreting islets are isolated from the pancreas and transplanted into diabetic patients. Since the Edmonton protocol was developed in 1999, about 900 cases of diabetes have been treated by allogeneic transplantation of islets in the United States and Europe. In recent years, the 5-year survival rate of transplanted islets has reached 50%, making islet transplantation an important alternative to diabetes treatment. However, this is the best alternative for the treatment of type 1 diabetes, but the biggest barrier is the lack of pancreas to be implanted. As an alternative to this, studies are currently under way to isolate islets from pigs and transplant them to humans. Piglet islets are physiologically similar to humans, can obtain large quantities, and have the advantage of facilitating genetic modification. The possibility of diabetes treatment using pancreatic islets has been demonstrated in several studies using rodents and primates. However, pre-clinical studies using primates should be preceded by the application of pig islets to humans to treat diabetes. Since 2005, Emory University in the United States and Minnesota University in the United States have reported the survival of transplanted islets over 6 months in 6 groups since the first identification of pig islet transplants in primates and long-term survival of transplanted pig islets. As a result, the heterologous organ donation community has confirmed the possibility of clinical application of porcine pancreatic islet transplantation, and international guidelines and guidelines for clinical application of xenogeneic pancreatic islets have been agreed and enacted through the 2009 International Society of Transplantation (IXA). According to these guidelines, the successful criteria for preclinical studies for clinical applications are that normoglycemia or minimal levels of porcine C-peptide are present in 8 of the pigs for at least 6 months after transplantation of the pig islet product into diabetic primates Lt; / RTI > However, studies that meet these international guidelines have not been conducted worldwide except for immunosuppression therapy.

The long-term survival of a transplanted islet depends on how effectively it suppresses the various immune responses to the transplanted islets. These immune responses can be divided into two types: instantaneous bloodmediated inflammatory reaction (IBMIR) that occurs within the transplantation moiety, immune response by T cells and B cells involved in acute and chronic rejection. The long-term survival of double-transplanted islets depends on how effectively T cells are regulated. In order to inhibit the immune response by T cells, drugs such as MMF, Rapamycin, cyclosporine, Tacrolimus, leflunomide, alemtuzmab, CTLA4-Ig, LFA-3-Ig, FTY720, Bortezomib and CD40-CD40L blockade have been studied. These agents and their combinations have been published with promising results in the treatment of diabetes mellitus by islets transplanted from rodents and primates. However, studies on primates that are essential for clinical applications have not yet published valid studies that meet the IXA guidelines for clinical applications. This implies that these immunosuppressive therapies can be selectively applied to higher animals and humans of primates or higher, without validated results. In addition, the combination of existing immunosuppressants means that short - term survival of the pancreas is possible by inhibition of T cells, but there is a limit in inducing long - term survival in vivo.

CD4 ⁺ regulatory T cells play a role in protecting the body from autoimmune diseases by controlling unnecessary immune activities in vivo and inducing immune tolerance. Recently, studies are underway to transplant control T cells in vitro and transplant them into animals with autoimmune diseases. In the case of pancreatic islet transplants, many studies on some rodents have been published, but there are no studies on primates. Accordingly, the present inventors have developed a co-administration method of immunoregulatory T cells and a CD40-CD40L binding inhibitor as a method for maintaining long-term survival of porcine pancreatic islets transplanted into primates with high reproducibility. It was also confirmed that this combination administration method can proliferate CD8 ⁺ Treg cells having other regulatory functions in addition to CD4 ⁺ regulatory T cells in vivo. Therefore, it was confirmed that the combined administration of the immunoregulatory T cells and the CD40-CD40L binding inhibitor remarkably alleviated the immune response upon organ transplantation and completed the present invention. When using this method, all 5 of the 5 streptozotocin-induced diabetic monkeys treated with the pig transplantation survived for 6 months or more, and 2 of them still maintained normal blood glucose without rejection, Over 500 days are alive for a long time. This is the world's first achievement as a result of satisfying all the criteria for clinical application of xenotransplantation as defined by the International Society of Xenotransplantation.

The present inventors have intensively studied a reproducible and consistent immunosuppressive method for xenotransplantation between primates and pigs. As a result, they have been transplanted into primate diabetic-primate monkeys and then immunoregulatory T cells and CD40-CD40L binding inhibitors The present inventors completed the present invention by confirming that all of them survived for at least 6 months or more for a long period of time.

Accordingly, it is an object of the present invention to provide a composition for inhibiting an immune response of a tissue graft receptor comprising an immunoregulatory T cell and a CD40-CD40L binding inhibitor, an immunosuppression method, and a tissue inhibited by the immune response.

According to one aspect of the present invention, there is provided an immunosuppressive composition comprising an immunomodulatory T cell and a CD40-CD40L binding inhibitor.

A major feature of the present invention is that the heterologous organs survived for the first time by inhibiting the immune response using immunoregulatory T cells and CD40-CD40L binding inhibitors.

The finding in the present invention is that the immune regulatory T cells and the CD40-CD40L binding inhibitor were used to proliferate another regulatory CD8 ⁺ CD28 ^- T cell having in vivo immunoregulatory function.

As used herein, the term " regulatory T cell "is a type of T cell that has the property of inhibiting the function of abnormally activated immune cells and controlling the inflammatory response, and is expressed as Treg . These immunoregulatory T cells can be divided into natural Treg and adaptive Treg cells. Peripheral T cells can be differentiated into cells that exhibit immunosuppressive effects when stimulated by a self or external antigen under specific circumstances. These cells are called adaptive Tregs or inducible Tregs, and Tr1, TGF and Th3 and CD8 Ts that secrete -beta.

Preferably, the immunoregulatory T cells of the present invention are not limited in their kind, but may be autologous, allogeneic or xenogeneic immunoregulatory T cells, most preferably autologous.

Immunoregulatory T cells are produced in the thymus and present in small amounts, but the process of isolating and culturing them is well known in the art. For example, in vitro generation of CD4 ⁺ CD25 ⁺ regulatory cells from murine naive T cells Nature Protocols 2 , 1789 - 1794 (2007), which is incorporated herein by reference.

Preferably, the immunoregulatory T cells of the invention have CD4 ⁺ CD25 ⁺ CD127 ^- label.

According to one embodiment of the invention, by ATG (Anti-thymoglobulin antibody) administration, and the immunomodulatory immunosuppression of the present invention, T cells, and co-administration of the CD40-CD40L binding inhibitors CD8 ⁺ CD28 in vivo ^- with a cover Other immunoregulatory T cells are proliferated.

These cells have the function of inhibiting the activation of CD4 ⁺ T cells and control the self-tolerance of the immune system. Thus, the in vivo proliferation of CD8 ⁺ CD28 ^- cells with immunomodulating function plays an important role in the survival of transplanted islets.

According to a preferred embodiment of the present invention, said immunoregulatory T cells are proliferated in vitro according to the following steps:

(a) separating PBMC (peripheral blood mononuclear cells) from primate blood;

(b) dividing the isolated PBMC into CD4 ⁺ T cells and CD4 ^- PBMC cells using an anti-CD4 antibody;

^{(c ') CD4 + CD25 high} CD127 using anti -CD25 antibody and wherein the antibody -CD127 PBMC- derived CD4 ⁺ T cells comprising: ^- a fraction and CD4 ⁺ CD25 ^high Treg;

(c ") irradiating the CD4 ^- PBMC with radioactivity;

(d) stimulating CD4 ⁺ Treg of step (c ') by the following combination;

CD4 ^- PBMC in the range of 1 to 20, CD4 ^- PBMC in the range of 1 to 20 for CD4 ⁺ Treg, IL-2 in the range of 100 to 2,000 units / ml, TGF-beta in the range of 1 to 10 ng / ml, Anti-CD28 antibody, and IL2 (interleukin2); and

(e) repeating the step (d) one to ten times at intervals of 5 to 10 days.

The term "CD40-CD40L binding inhibitor " as used herein refers to a CD154 (CD40L) blocker, which is any agent that binds to CD154 and inhibits or prevents binding to the opposite receptor (e.g., CD40) .

Preferably, the CD40-CD40L binding inhibitor of the present invention is an antibody, an aptamer, a protein, a compound or a nucleotide binding to said CD40 or CD40L, more preferably an antibody or a compound, and still more preferably an antibody, Particularly a monoclonal antibody (MAb), and most preferably an antibody having antigen-specific binding characteristics of a 5c8 MAb. In the present invention, an anti-CD154 monoclonal antibody is used.

Preferably, the compositions of the present invention may be used for tissue or organ transplant rejection reactions. That is, the composition of the present invention can be used to inhibit or reverse the rejection of a tissue graft by a graft receptor, to prolong survival or to preserve the function of a tissue grafted to the graft receptor, .

Successful transplantation requires overcoming the receptor's immune rejection response to the cell or organ to be transplanted. The main mediator of transplantation immune rejection is T cells, which are immunoreactive by T cell receptor recognition of major histocompatibility complex (MHC) expressed in the graft, .

The transplant receptor of the present invention is a primate, and the primate may be selected from the group consisting of higher primates such as a monkey, a chimpanzee, a gorilla, an orangutan, and a human, preferably a monkey, a human, a chimpanzee and an orangutan And most preferably a monkey or a human.

In addition, the receptor may be another mammal in need of tissue transplantation, particularly a mammal of commercial importance, or a pet or other valuable animal, such as a species at risk of becoming extinct. Thus, non-limiting examples of receptors include sheep, horses, cows, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice.

Preferably, the tissue of the invention is allogeneic to the primate or is xenogeneic to the primate.

The donor tissue may be derived from a donor or other live donor, or cadaver donor, by conventional means. The donor is preferably tissue compatible to be operable using the receptor. Thus, the receptor is preferably autologous and allogeneic donor tissue. However, the donor tissue may be obtained from a heterologous species, such as a non-human primate (e.g., chimpanzee or beet) or other relatively suitable animal (e.g., pig).

Preferably, the tissue is whole pancreatic tissue of the pig or isolated islets.

The composition of the present invention may also be used in combination with an anti-inflammatory agent such as ATG (anti-thymoglobulin), sirolimus, methylprednisolone, corticosteroids, antiproliferative agents, cyclosporine, tacrolimus, mycophenolate mopetil, mucorubin, deoxysperguanine, Nasal sodium, re flunomide, azaspirane, and combinations thereof, and may be variously combined depending on the situation.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating an autoimmune disease or inflammatory disease, which comprises an autologous-derived immunoregulatory T cell (Regulatory T cell) and a CD40-CD40L binding inhibitor as an active ingredient to provide.

As used herein, the term " autoimmune disease "refers to a problem in induction or continuous maintenance of self-tolerance, resulting in an immune response to a self-antigen and thereby attacking its own tissue. It means disease that occurs.

As used herein, the term "inflammatory disease" refers to a tumor necrosis factor-a (TNF-a) secreted from immune cells such as macrophages by overexpressing the human immune system due to harmful stimuli, Inflammatory substances (inflammatory cytokines) such as interleukin-1 (IL-6), prostagladin, luecotriene or nitric oxide (NO) .

Examples of such autoimmune diseases include, but are not limited to, alopecia greata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune adison disease, autoimmune diseases of the adrenal glands, autoimmune hemolytic anemia, autoimmune hepatitis, And celiac spruedermatitis, chronic fatigue immunodeficiency syndrome, chronic inflammatory dehydration polyneuropathy, Churg-Strauss syndrome, cicatricial hyperplasia, autoimmune thrombocytopenia, Behcet's disease, Idiopathic thrombocytopenia, idiopathic thrombocytopenia, idiopathic thrombocytopenia, idiopathic thrombocytopenia, idiopathic thrombocytopenia, idiopathic thrombocytopenia, idiopathic thrombocytopenia, idiopathic thrombocytopenic purpura IgA neuritis, inflammatory arthritis, squamous cell carcinoma, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis Atherosclerotic plaques, scleroderma, Type I or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus pemphigus, malignant anemia, polycythemia multiforme, polychondritis, autoimmune multinucleate syndrome, rheumatoid polyposis, multiple myositis and dermatomyositis, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment and / or prophylaxis of cirrhosis, psoriasis, psoriatic arthritis, Rheumatoid arthritis, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, But are not limited to, arthritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.

Preferably, autoimmune diseases that can be prevented or treated by the pharmaceutical composition of the present invention include rheumatoid arthritis, type 1 diabetes, multiple sclerosis, systemic lupus erythematosus and atopy.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally. When administered parenterally, the pharmaceutical composition of the present invention may be administered by intravenous infusion, subcutaneous infusion, muscle infusion, and intraperitoneal infusion. In the pharmaceutical composition of the present invention, the route of administration is preferably determined depending on the type of disease to which it is applied.

For example, when applied to Type 1 diabetes, peritoneal administration is most preferred because the administered immunoregulatory T cells and CD40-CD40L binding inhibitor can effectively migrate to the pancreas without dilution.

 The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . The dosage of the pharmaceutical composition of the present invention is preferably 0.5 to 100 mg / day / kg body weight.

Also, in the present invention, the immunoregulatory T cells contained in the composition are 1 x 10 ³ - 1 x 10 ¹² cells / kg and the CD40-CD40L binding inhibitor is 1 - 100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.

Since the composition of the present invention contains the self-derived immunoregulatory T cell (Regulatory T cell) and the CD40-CD40L binding inhibitor as an active ingredient, the composition of the present invention is not limited to The description thereof is omitted in order to avoid excessive complexity of the present specification.

According to another aspect of the present invention, the present invention provides a method of immunosuppression for tissue transplantation comprising administering to a subject an effective amount of a self-derived immunoregulatory T cell (Regulatory T cell) and a CD40-CD40L binding inhibitor to provide.

According to another aspect of the present invention, there is provided a method for the treatment of a subject, comprising the step of administering to a transplant recipient a subject, an ex vivo amplified self-derived immunomodulatory T cell, a CD40-CD40L binding inhibitor and an immunosuppressive agent A method for inhibiting an immune response.

In addition, the present method provides a tissue in which the immune response is suppressed, and preferably, the tissue is a whole pancreatic tissue of the pig or a separate islet.

Since the method of the present invention suppresses the immune response by using the self-derived immunoregulatory T cells (regulatory T cell) and the CD40-CD40L binding inhibitor, the above-described composition of the present invention is redundantly described The description thereof will be omitted in order to avoid excessive complexity of the present specification.

The present invention makes it possible to survive porcine pancreatic islets transplanted into primates for a long time and exhibits high reproducibility, so that it can be effectively used to overcome various immune rejection reactions occurring in recipients of allogeneic or xenogene organs .

Figure 1 shows the propagation efficiency according to each condition.
Figure 2 shows the results of subpopulations analysis of CD4-PBMCs that contribute to the mass proliferation of Tregs.
Figure 3 shows the characteristics of Tregs grown according to the two-parent propagation protocol.
Figure 4 shows the CD4 + T effector cell inhibitory effect of proliferating Tregs.
Figure 5 shows the phenotypic analysis results of the proliferated nTregs.
Fig. 6 schematically shows optimization conditions and methods of Treg proliferation.
Fig. 7 shows the results of analysis of the initial islet loss and transplanted beta- cell mass. (A) Pig C-peptide release at initial time-points (15 min, 1 hr and 4 hrs) after islet transplantation was assessed by early graft failure group (R037, R050, R063; these animals lost graft function within 10 days . Anti-CD154 monoclonal antibodies were not included in the immunosuppressive protocol) and in serum samples of the long-term graft survival group (R051, R080, R082, R084, R089). The difference in the level of C-peptide between the two groups at 15 min was statistically significant (p < 0.05), suggesting that the initial islet destruction of the long-term survival group is significantly smaller than in the initial graft failure group. (B) The ratio of pig C-peptide to fasting blood glucose (CP / G) was measured on the 14th day of the initial graft failure group (R005, R006, R013, R038, R050; these animals lost graft function within 14 days) And long-term survival group (R051, R080, R082, R084, R089). The difference in CP / G ratio between the two groups was very significant, suggesting that the transplanted beta- cells were significantly higher in the long-term survival group than in the early graft failure group.
Figure 8 shows the glucose control by transplanted piglet islets. Fasting blood glucose and pig and monkey C-peptide were measured in five monkeys transplanted with adult pig islets. R051 and R082 were insulin-independent for> 603 days and 513 days, respectively. R080, R084 and R089 were insulin-independent for 168, 303, and 180 days, respectively. The absence of the monkey C-peptide measured at intervals indicates that glycemic control is entirely dependent on the porcine C-peptide from the transplanted porcine islet rather than endogenous beta cell regeneration. Gray line: fasting blood sugar, red bar: pig C-peptide, inverted triangle (▼): monkey C-peptide, pink bar: exogenous insulin.
Figure 9 shows an intravenous glucose tolerance test and a pig C-peptide reaction. (A) Adult pig pancreatic islets were implanted in each monkey and IVGTT was performed at designated time-points. The designated glucose degradation rate (K _G ) at each time-point represents the ability of each monkey to have good glucose handling (K _G > 1) during normal blood glucose. (B) Pig C-peptide was negative before transplantation (DM), but C-peptide secretion of pigs was observed in all monkeys in response to glucose load. (C) The area under the curve (AUC) of the pig C-peptide released during IVGTT was calculated. A sustained AUC value higher than the DM state value suggested that the blood glucose concentration was controlled by the transplanted porcine islets.
Figure 10 shows changes in body weight (BW) of individual monkeys. All monkeys lost 20% of their body weight after STZ injection, but they maintained (R080, R082, R084, and R089) or increased (R051) after pig transplantation. R080 BW decreased temporarily in DPT 110 due to severe diarrhea, but recovered later. The BWs of R082 and R084 probably decreased from 150 days to 200 days due to anorexia due to the addition of new immunosuppressants.
FIG. 11 shows the results of monitoring Lethus CMV in peripheral blood of monkeys after transplantation of piglets. The copy numbers of plasma CMV (RhCMV) DNA in plasma were measured by quantitative real-time PCR and the antibodies were titrated by immunofluorescence analysis. Serially diluted serially from 1:10 were incubated with acetone-fixed RhCMV strain 68-1-infected HEL299 and incubated with FITC-conjugated anti-monkey IgG antibody for 30 minutes at room temperature. Antibody titration was defined as the highest serum dilution with bright fluorescence. Intermittent RhCMV viremia was observed in all monkeys, but clinical signs associated with disease and RhCVM reactivation were not observed. Importantly, the anti-RhCMV antibody titer was constantly increased with RhCMV viremia, suggesting a thorough humoral immune response to CMV reactivation.
Figure 12 shows the difference between CD8 ⁺ CD28 ^- T cells and CD8 ⁺ CD28 ⁺ T cells and the ratio of CD8 ⁺ CD28 ^- T cells / CD4 ⁺ T. (A) Absolute numbers of CD8 ⁺ CD28 ^- T cells, CD8 ⁺ CD28 ⁺ T cells and CD4 ⁺ T cells were generally monitored in peripheral blood for 1-2 times per week for up to 6 months, (Red line: CD8 ⁺ CD28 ^- T cells, blue line: CD8 ⁺ CD28 ⁺ T cells) until the graft rejection date of each monkey. Of T cells and CD8 ⁺ CD28 ⁺ T cells ^{- (DPT180 R051: DPT603, R080} : DPT180, R082: DPT537, R084:: DPT503, R089) to the measured each day CD8 ⁺ CD28 (B) from transplant day graft rejection day Absolute numbers were plotted. Monkeys CD8 ⁺ CD28 ^- T cells and Treg injection (Treg: R051, R080, R082 ) is, Treg injection without monkeys: significantly higher (Non-Treg R084, R089) (p <0.05, Analysis of Variance (ANOVA ) And the Tukey Post Hoc test using SPSS ^® statistical software version 21). Data are expressed as a mean ± SEM scatter plot on the red line. (C) The fasting glucose level after transplantation of pig islets was inversely proportional to the ratio of CD8 ⁺ CD28 ^- to CD4 ⁺ T cells in peripheral blood (red line: ratio of CD8 ⁺ CD28 ^- CD4 ⁺ T cells, gray line: Level (mg / dL)). A thick red bar represents the period of time that monkeys treat exogenous insulin. (D) PBMCs were obtained from monkeys at designated times, and CD4 ⁺ T, CD8 ⁺ CD28 ^- T and CD8 ⁺ CD28 ⁺ T cells were sorted and augmented by FACS Aria III. CD4 ⁺ T cells were labeled with CFSE and stimulated with anti-CD3 antibody and irradiated, self-derived antigen-presenting cells (APC) (CD4 ^- CD8 ^- PBMC) in the presence or absence of CD8 ⁺ CD28 ^- T cells for 5 days. Proliferation of CFSE-labeled CD4 ⁺ T cells was measured by FACS analysis. CD8 ⁺ CD28 ^- T cells from normal blood glucose animals (R051 and R082), but not the untreated monkey (R092), have an immunosuppressive effect on CD4 ⁺ T cells.
Figure 13 shows ELISPOT analysis and measurement of anti-Gal IgG levels. (A) IFN-γ and / or IL-17-secreting T cells were measured at intervals of 2 or 3 months and IFN-γ and IL-17 + T cell responses to most pig islets were inhibited.
(B) Plasma levels of anti-αGal IgG and IgM, anti-nonGal IgG and anti-donor-specific IgG were measured to evaluate the humoral immune response. Although these were almost stable, an intermittent increase in anti-Gal or non-Gal IgG levels was controlled by increasing the frequency of administration of the anti-CD154 monoclonal antibody every other week. Increased anti-αGal IgG and anti-donor-specific IgG recurred hyperglycemia in R082 and R084, suggesting a relationship between persistent rejection and humoral response.
Figure 14 shows immunohistochemical staining of liver biopsies. (A) were biopsied from R051, R080, R082, R084 and R089 in DPTs 560, 184, 296, 120 and 219, respectively. In order to identify pig beta cells, T cells, CD4 T cells, CD8 T cells, B cells and macrophages, liver samples were incubated with anti-insulin, CD3, CD4, CD8, CD20 and CD68 antibodies and the corresponding secondary antibodies And analyzed by immunohistochemical staining. Islet from normal blood glucose, R051 and R082, did not have immune cell infiltration such as T cells, B cells and macrophages while the other islets (R080, R084, R089) were destroyed by infiltrated CD8 ⁺ T cells . (B) The degree of T cell infiltration into pig islets was semi-quantitatively analyzed using a histopathological grading system (grade 1, no infiltration, grade 2, peri-islet infiltration, grade 3, Intra-islet infiltration, and quaternary; total destructive infiltration. Hyperglycemic animals (R080 and R089) exhibited combined tertiary and quaternary ratios of> 50%.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the appended claims. It will be obvious to you.

Materials and Methods

animal

Five Macracamulatta monkeys aged 3-4 years were used for the study. After importing from China, it was confirmed that all monkeys were in good condition after quarantine for one month. After that, the monkeys were acclimated to the animal facility for more than six months. Monkeys in individual cages were fed diets (PS DITE® fresh fruit, and vegetables; Oriental Yeast Co. Ltd., Tosu, Japan) and water. The environmental conditions in the breeding facility maintained a 12-hour light-dark cycle (beginning at 7:00 am), 13-18 hours of ventilation, 24 ± 4 ° C and 50 ± 10% relative humidity. The pig, an islet of donor, was crossed and isolated by Seoul National University (SNU) mini pig at designated pathogen-free (DPF) facility. In addition, microbiological tests were conducted on 41 viral pathogens including PERV (porcine endogenous retrovirus), 35 bacterial pathogens, 2 fungal pathogens and 25 parasites. Of the 12 SNU miniature pigs in this study, 4 were female (mating experience, N = 2; no mating experience, N = 2). The average age of 12 pigs was 36.5 months [19-91 months]. The procedure for all animal experiments was conducted under the approval of the IACUC (IACUC no. 12-0374-C1A1) at Seoul National University Hospital.

Isolation of pig islets

All SNU miniature pig pancreatectomies were performed in a sterile operating room. Surgical procedures during the study period were performed in the same manner as known methods. After transfection, pancreatic islet isolation was performed using a known modified Ricordi method. In summary, the obtained pancreas was expanded in the conduit using a preservation solution containing Liberase MTF C / T (Roche Diagnostics). The average neutral protease / collagenase (NP / C) ratio for separation experiments of each islet was 39.0 neutral protease active units per Wunsch unit.

Inducing diabetes in primates

After the monkeys were anesthetized, a 5-Fr (Dual-Lumen PICC; Bard Access System) was inserted into the right internal jugular vein of the monkey. Monkeys were fasted for one day and renal toxicity was reduced by infusing saline (0.9% NaCl, 40-60 ml / kg / day iv) via a tether system for 12 hours before STZ (USB Co.) administration. VUTORPANOL or methoclopramide was also administered to prevent vomiting by STZ. High-dose STZ (110-120 mg / kg) was diluted in 10 ml saline and administered intravenously for 5 minutes.

IVGTT (Intravenous glucose tolerance test)

Additional saline was infused for 2-4 hours. Glucose levels in the blood were measured using a portable blood glucose meter (Accu-Chek) or a continuous blood glucose monitoring system (Guardian RT; Medtronic Inc.). Monkeys were given STZ in the morning and provided light meals in the afternoon. In the case of loss of appetite, dextrose was injected through the tetra system to prevent hypoglycemia. Liver and kidney function tests were performed before and after STZ administration, and c-peptide levels were measured using a radioimmunoassay (Linco) according to the manufacturer's protocol. After successful induction of diabetes, glucose levels in the blood were measured at least twice or three times a day. Hyperglycemia was controlled by subcutaneous injection of human recombinant insulin such as Humalog (Eli Lilly), Novolin N (Green cross), or Lantus (Sanofi-Aventis). Fasting and nonfasting blood glucose levels in diabetic monkeys were maintained at approximately <150 mg / dl (> 8.3 mmol / l) and <200 mg / dl (11.1 mmol / l), respectively. Complete diabetes was identified with persistent hyperglycemia based on venous glucose tolerance test and c-peptide levels of 0.2 ng / ml. Briefly, after fasting for one day without insulin, 0.5 g / kg of a 50% dextrose solution with the same volume of saline was intravenously injected for 1 minute. Glucose levels in monkey blood were measured before injection and at 2, 5, 15, 30, 60, 90, and 120 minutes after injection. Insulin and c-peptide levels were measured at the same time intervals. The glucose degradation rate (K _G ) represents the log-linear (ln) reduction of the glucose level between IVGTT 5 and 30 minutes and was calculated using the following equation:

KG = ln (glucose level at 5 min) - ln (glucose level at 30 min) / 25

Porcine islet transplantation into primate (NHP)

All monkeys were fasted 12 hours prior to surgery. Monkeys were pretreated with atropine sulfate (0.04 mg / kg sc; Huons), anesthetized with thiopental Na (25 mg / kg iv; JWP) and treated with 1% isoflurane ), N ₂ O and O ₂ . During the operation, Lactated Ringer's solution or 5% dextrose saline was intravenously injected. (25 mg / kg iv; Chong Kun Dang) was injected intravenously, and meloxicam (0.2 mg / kg iv; Boehringer Ingelheim) was administered before surgery to prevent inflammation and analgesic effects. Respectively. Laparotomy was performed to expose the jejunal arch, and the islets were injected. A 24- or 22-gauge catheter was inserted through the plant vein and approached near the portal vein. The pig pancreatic islets were injected with gravity for 8-12 minutes. After injection, blood vessels were ligated with 5-fluorene sutures. Finally, the abdominal cavity was sutured by a general method. After the operation, a tether system was applied for continuous fluid therapy and low dose sugar infusion if needed.

Immunosuppressive therapy

The immunosuppressive therapy used in the present invention is based on anti-CD154 monoclonal antibody (5C8, National Institutes of Health, Bethesda, MD) and immunoregulatory T cells. In addition, immunosuppression was induced including conventional immunosuppressants such as sirolimus (Rapamune (R) Wyeth, Philadelphia, PA) and anti-thymocyte globulin (ATG, Thymoglobulin (R) Genzyme, Cambridge, 20 mg / kg of anti-CD154 monoclonal antibody was injected intravenously at the transplantation-10, -7, -4, 0, 3, 7, and 12 days and then injected once a week for 3 months. Oral doses of rapamycin from 3 to 8 ng / ml were stabilized daily. ATG was administered at transplantation-3 and -1 days. CVF (Cobra venom factor, 100 U / kg, Quidel, San Diego, Calif.) Was administered at transplantation-1 to prevent complement activation. TNF-α neutralizing monoclonal antibody adalimumab (Humira ^® , Abbott Laboratories Ltd., Queenborough, UK) was subcutaneously administered to the recipient monkey at a dose of 5 mg / kg 2 - 3 hours before the islet injection.

Isolation and proliferation of immunoregulatory T cells (Treg)

PBMC (Peripheral blood mononuclear cells) were isolated from 20 ml of blood collected using Pycor-Fake ™ (1.077 g / dl; GE Healthcare Life Science, Pittsburgh, PA, USA). CD4 ⁺ T cells and CD4 ⁺ T-depleted cells (CD4 ^- PBMC, CD4 ^- T) were isolated using NHP CD4 ⁺ T - cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) and autoMACs ® according to the manufacturer 's (Miltenyi Biotec) Cells).

CD4 ⁺ T cells were stained with the following conjugated mAbs: CD4-FITC (OKT4; BD Bioscience, San Jose, CA, USA), CD25-PE (CD25-4E3; eBioscience, San Diego, CD127-PEcy7 (R34.34, eBioscience). Anti-CD25-PE (BC96, BD Bioscience) was also used to test flow cytometry of CD4 ⁺ CD25 ^high CD127 ^- T cells.

A portion of CD4 ^- PBMC was used as a stimulant cell for subsequent Treg proliferation and the remainder was stored frozen for the next two rounds of growth after 7 days.

About 1-2% in CD4 ⁺ T cells using FACSAria ^™ III Cell Sorter (BD Bioscience) to isolate CD4 ⁺ T cells (designated CD25 ^high ) expressing about 90% FoxP3 and very high CD25 Of CD25 ^high CD127 ^- CD4 ⁺ Treg were collected. At the same time, CD4 ⁺ CD25 ^- CD127 ⁺ T cells were separately collected and cryopreserved for subsequent CFSE (carboxy fluorescein succinimidyl ester) -based T cell inhibition assays. Treg groups of 95-99% purity were obtained from flow cytometry. Per well ^{CD4 + CD25 high CD127 - Treg 1x10} 4 cells and 1x10 ⁵ cells of irradiated, self-derived CD4 ^- PBMCs (APC, antigen-presenting cells) were seeded in 96-well plates (Nest ^® , CA, USA) coated with anti-CD3 antibody (FN18, 5 ug / ml; BD Bioscience) USA).

(RhTGF-beta, 5 ng / ml; PeproTech, Rocky Hill, NJ, USA) and recombinant human IL-2 (rhIL-2, 500 units / ml; Chiron, Emeryville, The cells were cultured for the first 3 days with anti-CD28 antibody (CD28.2, 5 / / ml; e-Bioscience). On days 3 and 5, two-thirds of the medium was replaced with fresh medium containing rhIL-2 (1,000 units / ml) and rhTGF-beta (5 ng / ml). After 7 days of collection, the cells were re-stimulated and cultured for an additional 7 days with the same protocol as described above. The cryopreserved CD4 ^- PBMCs were thawed, cleaned and irradiated to supply APCs during the second proliferation. If necessary, PBMCs were further separated from fresh blood and CD4 ^- PBMCs were obtained using an autoMACs cell isolator. Finally, the proliferated Tregs were collected and purified by Ficoll-Hypaque density gradient centrifugation.

Cells were cultured in X-VIVO 15 medium (BioWhitaker, Walkersville, MD, USA) supplemented with 10% fetal bovine serum, 2-mercaptoethanol, L-glutamine and non- 5% CO ₂ . Foxp3 expression levels of proliferating Tregs were stained with anti-CD4-FITC, anti-CD25-PerCP, and anti-FoxP3-PE (PCH101, eBioscience) and analyzed using flow cytometry.

Control T cells that have been cultured for a total of 15 days are pooled in PBS buffer and injected through the blood vessels of primates.

CD154 monoclonal antibody (5C8, National Institutes of Health, Bethesda, MD) and immunoregulatory T cells (Treg) for each diabetic monkey individual as shown in Table 1 below, or anti-CD154 monoclonal antibody alone . R092 did not transplant any treatment as a normal control. Expression levels of FoxP3 in proliferating Tregs were analyzed by flow cytometry by staining with anti-CD4-FITC, anti-CD25-PerCP, and anti-FoxP3-PE (PCH101, eBioscience).

R051 R080 R084 R082 R089 R092 CD40-CD40L
blockade + + + + + - The proliferated Treg + + - + - - Other immunosuppressants + + + + + -

Functional monitoring of the graft

Glucose levels in the blood of the recipient were measured using a portable blood glucose meter (Accu-Chek) or continuous blood glucose monitoring system (Guardian RT; Medtronic Inc.). Pork c-peptide levels in the blood of the recipient were measured twice a week by radioimmunoassay (LINCO research Inc., St. Charles, Mo.). Failure of the grafts was confirmed by the absence of porcine c-peptide and the need for exogenous insulin to control hyperglycemia.

CD8 ⁺ CD28 ^- T cell suppressor assay

CD8 ⁺ CD28 ^- T cells and CD4 ⁺ T cells were isolated from the blood of R051 and non - transplanted normal monkey R082 in order to confirm the immunoregulatory function of the in vivo proliferated CD8 ⁺ CD28 ^- T cells. CD4 ⁺ T cells were labeled with the fluorescent marker CFSE (carboxyfluorescein diacetate succinimidyl ester).

The CFSE-labeled CD4 ⁺ T cells (5 x 10 ⁴ cells / well) were irradiated in the respective ratios (1: 1 to 1: 8) in the presence of FACSAriaIII-isolated autologous CD8 ⁺ CD28 ^- T cells - Stimulated with 96-well plates coated with anti-CD3 antibody with self-derived APCs (CD4 ^- CD8 ^- PBMCs). After 5 days of culture, proliferation was evaluated by FACS staining with anti-CD3 antibody and anti-CD4 antibody. CFSE-labeled CD8 ⁺ CD28 ^- T and CD8 ⁺ CD28 ⁺ T cells were treated with anti-CD3 antibody and irradiated-self-derived APCs (CD4 ^- CD8 ^- PBMCs) for 5 days to assess T cell immune deficiency Stimulation, and proliferation was analyzed by FACS Canto II. That is, how much the CD8 ⁺ CD28 ^- T cells added at the respective ratios inhibited the proliferation of CFSE-labeled CD4 ⁺ T cells was confirmed by flow cytometry.

ELISPOT (Enzyme-linked immunosorbent spot) assay

ELISPOT analysis was performed by a known method. Briefly, the frequency of IFN-y or IL-17A-secreted antigen-specific T cells in peripheral blood of non-human primates (NHP) was measured using an ELISPOT kit (Mabtech). The resulting spots were counted on a computer-assisted ELISPOT reader system (AID).

Measurement of anti-Gal IgG levels

As known in the art, the plasma concentration of anti-Gal IgG was determined using an enzyme-linked immunosorbent assay (ELISA).

Flow cytometry for anti-non-Gal antibodies

As is well known in the art, the use of α-1,3-galactosyltransferase gene knockout (GTKO) endothelial cell lines (PEC69) Gal antibody responses were evaluated by flow cytometry.

Quantification of RhCMV (rhesus cytomegalovirus)

Quantification of RhCMV was confirmed using the following oligonucleotides and real time PCR: forward primer 5'- ACAGAGGCCAGTGGGATGTC-3 ', reverse primer 5'-CCCTGATGATGGGCATAGATAAG-3'; And probe 5 'FAM- CCAGGCACATTCTCTGGGAGCACAC-3' TAMRA (Bioneer Co.). These sequences are highly conserved in the RhCMV 156 region and specific for RhCMV IE2.

Antibody measurement

IgG anti-RhCMV antibody titers were measured by indirect immunofluorescent staining.

Biopsy and Immunohistochemistry

After the anesthesia, the monkey was laid down and the skin and abdominal wall were incised from the dentate to the navel. The hepatic margin was carefully held and about 10 mm distal to the border was excised. Hemorrhage at the site of resection of the biopsy was controlled by electrocautery and a hemostatic agent (SURGICEL® Johnson & Johnson Medical Korea, Seoul, Korea). The abdominal wall was sutured in the usual way. Liver tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. The embedded tissues were cut to a thickness of 4 탆 using a microtome. Paraffins were removed from the sections using xylene and rehydrated with ethanol at different concentrations. For the antigen retrieval, the prepared slides were treated by heat-induced antigen retrieval (HIER) using Tris-EDTA pH 9.0 solution and then washed with TBST (Tris-buffered saline with Tween 20) Respectively. The treated samples were incubated in a peroxidase blocking solution (DAKO, Denmark) and a protein block solution (Thermo Scientific, CA, USA), followed by a MultiVision polymer detection system , Thermo scientific, CA). Each section was incubated with primary antibody cocktail [insulin (Santa Cruz Biotechnologies, Inc., CA, USA) and CD3 (DAKO, Denmark), insulin and CD4 (Santa Cruz, CA, US), or insulin and CD8 UK) and washed four times with TBST. After staining, all stained slides were dried at 60 ° C and mounted with mounting solution (Richard Allan scientific mounting medium, Thermo Scientific, CA, US). The dyed samples were observed with a Zeiss Axio microscope (Germany) and the images were analyzed using Axiovision Rel. 4.8 (Zeiss, Germany).

Histopathological grade

Biopsied liver samples from monkeys were blotted onto paraffin and cut into 4-μm thickness. Immunohistochemical staining was performed using anti-insulin and anti-CD3 antibodies in tissue sections. The antigen - recovered islets were classified according to the degree of T cell infiltration. 1st grade; No invasion, grade 2; Islet-peri-islet infiltration, grade 3; Islet-intra-islet infiltration, grade 4; Total destruction infiltration. The number of at least 30 islets in all animals was analyzed using different regions (at least 1 μm apart in the previous section).

Statistical analysis

Statistical software GRAPHPAD PRISM5 (GraphPad Software, Inc., La Jolla, Calif.) Was used for the Mann-Whittney test (two-tailed P-value) and SPSS statistical software version 21 was analyzed using ANOVA (Analysis of Variance) and Tukey Post Hoc test Lt; / RTI &gt;

Experimental Example 1. Isolation and Expansion of Immune Regulatory T Cells (Treg)

We included rhIL-2 (500 units / ml) and TGF-beta (2.5 ng / ml) supplementation and TCR stimulation in plates with anti-CD3 antibody and free-formed anti- 3-fold propagation protocol was designed. In addition, irradiated CD4 ^- PBMCs were supplemented in this protocol as APCs. Due to technical problems in providing large amounts of freshly isolated Treg from blood, we firstly proliferated CD4 ⁺ CD25 ^high CD127 ^- Treg for one week under the conditions described above. After providing a sufficient amount of proliferating Tregs, different concentrations of rhIL-2 (500 and 1,000 units / ml) and TGF-beta (0, 2.5, and 5 ng / ml) and a variety of Tregs versus APCs The secondary and tertiary proliferation was performed in various combinations of culturing conditions including ratios (1: 0, 1: 2, 1: 4 and 1:10). After the second proliferation, the proliferation rate (various secondary and tertiary proliferation rates) and intracellular FoxP3 expression levels were compared. As shown in Fig. 1, Tregs were further proliferated at a concentration of 1,000 units / ml at a concentration of rhIL-2 of 500 units / ml. FoxP3 expression levels between the two groups were not significantly different. Tested at concentrations ranging from 0 ng / ml to 5 ng / ml, TGF-beta concentrations did not affect the proliferation efficiency of Treg, but higher levels of FoxP3 expression were present at higher concentrations. Interestingly, the proliferation rate was mainly influenced by the supplemental addition of CD4 ^- PBMC, an APC. A high proportion of CD4 ^- PBMCs versus Tregs resulted in a high proliferation rate of Treg while maintaining FoxP3 expression levels.

Taken together, we have found that the combination of 1,000 units / ml of rhIL-2, 5 ng / ml of TGF-beta and 1:10 ratio of Treg versus CD4 ^- PBMC inhibits FoxP3 expression levels of monkey Treg in vivo And it was confirmed that it was the optimum condition for growth.

Experimental Example 2: Monocyte as a major group enhancing NHP Treg proliferation

Next, CD8 ⁺ T, NK (CD16 ⁺ ), monocyte (CD14 ⁺ ) and B (CD20 ⁺ ) cells were transfected with FACSAria III to confirm the subpopulations of CD4 ^- PBMCs that contribute to the mass proliferation of Treg Were isolated from CD4 ^- PBMCs. The newly isolated CD4 ⁺ CD25 ^high CD127 ^- Tregs were subjected to primary proliferation for one week in the same manner as described above. Each separated subgroup was replaced with CD4 ^- PBMCs. As shown in FIG. 2, it has been found that monocytes play a major role in enhancing Treg proliferation. FoxP3 expression levels were also maintained in the presence of monocytes. However, CD8 ⁺ T, B and NK cells affected the proliferation at low levels.

Experimental Example 3. NHP CD4 ⁺ Establish a two-state propagation protocol for Treg proliferation

Finally, the inventors designed a second stimulating two-state NHP Treg proliferation protocol. As described above in Experimental Methods and Materials, isolated CD4 ⁺ CD25 ^hi CD127 ^- T cells were cultured for 3 days under stimulation with anti-CD3 antibody and free-formed anti-CD28 antibody conjugated plate were cultured with irradiated CD4 ^- PBMCs (Treg: APC = 1: 10) in the presence of rhIL-2 (500 units / ml) and rhTGF-β (5 ng / ml). On day 3 and 5, 2/3 of the medium was replaced with fresh medium containing rhIL-2 (1,000 units / ml) and rhTGF-beta (5 ng / ml). Seven days after the culturing, the collected Tregs were subjected to secondary proliferation repeatedly in the same manner as the primary proliferation. As shown in Fig. 9, the overall growth rate obtained from twelve runs of 9 monkeys during the two-week propagation period amounts to about 3,000 to 5,500 times. The proliferation rate was significantly retarded during the second proliferation period compared to the first proliferation period. The average Foxp3 expression level was about 92% (Figure 3c). The proliferating Treg consists of 99.9% CD4 ⁺ Tregs of 7AAD ⁺ live cells. Negligible amounts of live CD8 ⁺ T, monocytes, NK or or B cell cells were found after the final proliferation (FIG. 3D). For reference, the proliferation rate was delayed by an average of 2.35 times during the sole third-stage proliferation, even though the overall proliferation rate was increased by up to 12,500 times with additional tertiary proliferation. With these drawbacks, it was technically impossible to obtain a sufficient amount of CD4 ^- PBMCs during the third propagation period due to limitations of monkey 's collectable blood volume. Therefore, the present inventors finally established a second-stimulated 14-day NHP Treg proliferation protocol.

Example 4. Identification of strong immunosuppressive activity of proliferated Tregs

To confirm the immunosuppressive function of proliferating Tregs, CFSE-based T cell proliferation / inhibition assays were performed. CFSE- labeled autologous responder (responder) CD4 ⁺ CD25 ^- CD127 ⁺ T cells (effector T cell) of, Treg (CD4 ⁺ CD25 ^high CD127 ^-) 1 with respect to: the combination with 1 to 1:32 ratio, for 5 days CD3 antibody (0.5 [mu] g / ml) was conjugated with irradiated autologous CD4 ^- PBMCs (CD4 ⁺ T-depleted APC). As shown in FIG. 4, the proliferated Tregs showed a strong immunosuppressive effect on responder CD4 ⁺ T cell proliferation in a concentration-dependent manner. There were individual differences in the functional ability to inhibit autologous CD4 ⁺ T effector cells.

That is, 0: 1 (fitting not regulatory T cells: CD4 ⁺ CD25 ^- T) in three days 55% of the CD4 ⁺ CD25 ^- the proliferation of T cells made of, 1: 1 (regulatory T cells: CD4 + CD25-T cells ), 18.3% of CD4 ⁺ CD25 ^- T cells were proliferated for 3 days, indicating that regulatory T cells inhibited the proliferation of CD4 ⁺ CD25 ^- effector T cells.

Experimental Example 5: ⁺ Identification of Treg's phenotype profile

In the present invention, the activation state, mobility and expression of some function-related markers of the proliferated Treg were confirmed.

As shown in FIG. 5, in the phenotypic analysis, almost all the Tregs proliferated in this study expressed the active marker CD25 and the active and memory cell marker CD95. Significant amounts of CD69, CCR7 and PD-1 were expressed. Small amounts of CCR5 and CXCR3 were expressed in the proliferated Tregs. After proliferation of human CD4 ⁺ CD25 ⁺ cells, nearly all cells functionally expressed intracellular CTLA4, an important functional marker of CD27 and Treg, known to discriminate between regulatory and non-regulatory T cells. However, Tregs expressed negligible amounts of CD95L and intracellular IL-17 and IL-1. A small amount of IFN-y-secreting cells (1.8%) occurred during Treg proliferation. Overall, the Tregs proliferated in this study were identified as CD25 ⁺ CD95 ⁺ CD27 ⁺ CD47 ⁺ CTLA4 ⁺ FoxP3 ⁺ CD4 ⁺ Treg. In addition, the proliferated nTregs of the present invention did not express the inflammatory cytokines IL-17, IFN-r, and IL-10.

Experimental Example 6. NHP Treg proliferation protocol for preclinical experiments using monkeys

The method of isolating and propagating Treg is summarized in FIG. Generally, about 2-8 × 10 ⁷ PBMCs were obtained from 20 μl of monkey peripheral blood. CD4 ⁺ T and CD4 ^- PBMCs were separated by flow cytometry, and CD4 ⁺ CD25 ^high CD127 ^- Treg of about 1.5-3.5 × 10 ⁵ was concentrated and subsequent 14 - day proliferation was performed. The remaining CD4 ^- PBMCs were stored frozen for secondary proliferation. Treg growth rates for primary stimulation typically range from 3,000 to 5,500 times during the 14-day growth period. A proliferated Treg of about 4.5-17.5 × 10 ⁸ can theoretically be obtained from 20 μl of monkey peripheral blood. For reference, it is necessary in this protocol to collect peripheral blood from the same monkey in order to ensure a sufficient amount of CD4-PBMC as APC before the second proliferation in the total proliferation of nTreg.

That is, in the present invention, by using the self-derived APC and optimizing the conditions of anti-CD3, anti-CD28 antibody, IL-2 and TGF- ?, the propagation efficiency could be increased to 3000-5500 times on average over 14 days. In addition, the addition of regulatory T cells showed the effect of suppressing proliferation of CD4 + CD25-effector T cells and inflammatory cytokines.

Example 1. Identification of Piglet Islet Characteristics

The mass of the islets used for intraportal islet transplantation during allotransplantation in human clinical trials is 10,000 IEQ / kg of the receptor, but between 10,000 and 100,000 IEQ / kg of the receptor in various heterologous organ transplantation experiments between pigs and primates, kg. Since a large amount of pig islet is known to be lost soon after transplantation by a severe instant blood-mediated inflammatory reaction (IBMIR), we found that a sufficient amount of islets (~ 100,000 IEQ / kg) Were used to improve the engraftment of pig islets. Thus, the present inventors isolated swine pancreatic islets from two or three SNU miniature pigs at a time, collecting them at once as shown in Table 2, and injecting them into diabetic rhesus monkeys (Table 2).

The remaining 4 rats, except for the R051 monkey, were injected with 100,000 IEQ / kg of 430,000-540,000 IEQ, which is the total pancreatic islets. At this time, a small amount of an islet (15,000 IEQ) set was prepared to measure the quality of an islet. The present inventors used a dithizone staining method to measure the islet purity, TMRE, and FACS using Flouzin- Survival and diabetic NOD / SCID bioassay were used to assess the quality of the islets. From these quality assays we have found that the pig islets of the invention are highly pure (> 90% pancreatic islets), have a high viability (> 80% normal β-cells) and excellent diabetic therapeutic potential (> 60% A diabetic animal treated by the same animal).

Example 2. Confirmation of the therapeutic effect of the receptor monkey on diabetes after islet transplantation

2-1. Immunosuppression based on the anti-CD154 monoclonal antibody is effective in preserving islet chunks and controlling diabetes

The transplantation of pig islets into diabetic monkeys and the combination of immunosuppressive therapies confirmed that diabetes could be treated by the transplanted islets as a result of changes in blood glucose and the presence of pig c-peptide.

The present invention utilizes anti-CD154 monoclonal antibody-based immunosuppression for successful long-term survival and diabetes control of primate-transplanted porcine pancreatic islets.

In summary, anti-thymoglobulin (ATG) was used for induction, chimeric anti-CD154 monoclonal antibody and low dose of sirolimus as maintenance therapy.

When the receptor showed side effects of sirolimus, such as diarrhea or erythema, methylprednisolone (0.3 mg / kg) was used instead of sirolimus until sirolimus disappeared and then changed to sirolimus. In addition, CVF (cobra venom factor) and anti-TNFa (tumor necrosis factor-alpha) mAbs were used to reduce the initial loss of implant-pancreatic islets caused by IBMIR. Indeed, the islet breakdown was measured due to C-peptide release early (<4 hours) after transplantation (Fig. 7a) and the implanted mass was assessed due to the proportion of pig C-peptide to fasting blood glucose concentration within one month (Fig. 7b), indicating that the protocol of the present invention is able to protect the graft from initial loss and acute rejection rejection by IBMIR.

In the case of R051, R080 and R082, proliferated immunoregulatory T cells (Treg) were injected as follows: 1.87x10 ⁶ Treg cells at 6 days after transplantation in case of R051, 6 days after transplantation in case of R080 2.56 × 10 ⁷ and 2.73 × 10 ⁷ Treg cells on day 16 and day 16, and 6.20 × 10 ⁷ and 8.8 × 10 ⁶ Treg cells on day ⁶ and day 20 after transplantation, respectively.

When STZ-induced diabetic monkeys were transplanted with a sufficient amount of islets, the glucose concentration in the blood was rapidly reduced in all five mice, and blood glucose was excellently controlled for six months. During the entire follow-up period (6-23 months), pig C-peptide remained at high concentration (0.4-2 ng / ml), even after a long period of time. Remarkably, no monkey C-peptide was detected, suggesting that blood glucose is completely regulated by the transplanted porcine pancreas rather than the endogenous pancreatic islet of the receptor (Fig. 8).

In IVGTT results at regular intervals (1 ~ 2 months), animals showed constant glucose regulation ability in response to glucose injected by transplanting time. Over time, blood sugar levels were lower than at baseline. A significant amount of C-peptide was detected during IVGTT, but its level also decreased over time (Figure 9).

2-2. The receptor monkey was healthy and the immune system was not compromised

Importantly, there were no signs of serious side effects associated with the immunosuppression protocol. In particular, all monkeys were healthy and stable (R080, R084, R082 and R089) or showed a progressive weight gain (R051) (Fig. 10). One of the most important results of this experiment is that there is no indication of any side effects associated with transplanted piglet islets. Specifically, during the follow-up period, all monkeys showed stable bio-signals in terms of weight gain, normal appetite, physical activity, and body temperature, heart rate, respiration, etc. and maintained good health status.

In addition, real-time PCR analysis using specific primers for RhCMV (rhesus monkey cytomegalovirus) and anti-RhCMV antibody measurement by immunofluorescence analysis showed intermittent CMV viremia with an increased anti-RhCMV antibody, , But the CMV-related characteristics of the disease were unclear (Fig. 11). Valganciclovir was given to all monkeys during the initial 4 weeks post-transplantation (DPT1 to DPT28) for CMV, and these results are consistent with the findings of the present invention, as evidenced by the increase in RhCMV antibodies at RhCMV viremia Indicating that immunosuppressive therapy did not impair pathogen-specific humoral and T cell-mediated immunity. Overall, these results indicate that ATG induction combined with the chimeric anti-CD154 monoclonal antibody and the low-dose cyrolimus provided for long-term control of diabetes at the time of pancreas transplantation from pig to NHP would either cause side effects or impair pathogen-specific immunity Emphasize that it does not.

Example 3. CD8 ⁺ CD28 ^- Confirmation of proliferation of immunoregulatory T cells

The immunosuppressive therapy of the present invention confirmed that CD8 ⁺ CD28 ^- T cells proliferate in vivo according to the time after transplantation. As shown in FIG. 12, the amount of CD8 ⁺ CD28 ^- T cells known as regulatory T cells with immunomodulatory and inhibitory functions increased rapidly with time. CD8 ⁺ CD28 ^- T cells were isolated from the blood of monkeys and normal controls in combination with CD4 ⁺ Treg and CD40 - CD40L inhibitors to confirm immunoregulatory ability of CD8 ⁺ CD28 ^- T cells proliferating in vivo And the proliferation inhibitory ability of CD4 ⁺ T cells was confirmed. Immunosuppressive ability of CD8 ⁺ CD28 ^- T cells on CD4 ⁺ T cells in R051 monkeys with good glycemic control and R092 monkeys with no transplantation control was determined by concentration. As a result, in R051 monkeys, CD8 ⁺ CD28 ^- T cells inhibited CD4 ⁺ T cell activity by concentration. On the other hand, in R092 monkeys not treated with transplants and immunosuppressants, CD8 ⁺ CD28 ^- T cells were not immunosuppressed.

These results indicate that the use of the immunosuppressive therapy of the present invention promotes the proliferation of CD8 ⁺ CD28 ^- suppressive T cells in vivo.

On the other hand, IFN-y and / or IL-17-secreting T cells were measured by ELISPOT analysis at intervals of 2 or 3 months, as shown in Fig. 13A. During the observation period, IFN-y and IL-17 ⁺ T cell responses to porcine islets were inhibited, except for R080 (DPT205), where IL-17 ⁺ spots appeared to be increased due to diarrhea associated with microbial infection.

Also, as shown in Figure 13b, anti-αGal IgG and IgM, anti-nonGal IgG and anti-donor-specific IgG were used to monitor humoral immune response Were assessed weekly using ELISA and flow cytometry. Although these were almost stable, an intermittent increase in anti-Gal or non-Gal IgG levels was controlled by increasing the frequency of administration of the anti-CD154 monoclonal antibody every other week. Increased anti-αGal IgG and anti-donor-specific IgG recurred hyperglycemia in R082 and R084, suggesting a relationship between persistent rejection and humoral response.

Example 4. Confirmation of Chronic Rejection of Swine Islet by T Cells

In the case of R080 monkeys, the glucose level in fasting blood at 166 days after transplantation increased from 100 to 126 mg / dL. Despite the fact that exogenous insulin was actively administered, the glycemic control was slowly deteriorated at day 166, (Fasting blood glucose level, 260 mg / dL).

To investigate what happened at the site of the islet transplantation, biopsies were performed at the liver end (2 cm wide, 1 cm high) 184 days after transplantation.

As a result of extensive immunohistochemistry using insulin, CD3, CD4, and CD8 antibodies to the biopsy samples, the b-cells of pig islets were strongly stained with insulin, but these cells were infiltrated by CD8 ⁺ T cells (Fig. 14). Interestingly, CD4 T cells were found to be located at the distal side and did not appear to penetrate into the pancreatic islets.

Abbreviation

CFSE; Carboxyfluorescein succinimidyl ester, CVF; Cobra venom factor, CMV; cytomegalovirus, DPT; Day post transplantation, ELISPOT; Enzyme-linked immunoSpot, IBMIR; Instant blood-mediated inflammatory reaction, IXA; International Xenotransplantation Association, Monoclonal antibody; mAb, NHP; Nonhuman primate, NOD / SCID; Non-obese diabetic and severe combined immunodeficiency, PEC; Porcine endothelial cells, PERV; Porcine endogenous retrovirus, SNU; Seoul National University, STZ; Streptozotocin, TNF; Tumor necrosis factor, T1D; Type 1 diabetes

Claims (13)

Immunoregulatory T cells derived from primates and ex vivo (Regulatory T cell, Treg); Anti-CD154 monoclonal antibody which is a CD40-CD40L binding inhibitor; And an immunosuppressive agent,
Said immunoregulatory T cells comprising (a) separating PBMC (peripheral blood mononuclear cells) from the blood of a primate;
(b) dividing the isolated PBMC into CD4 ⁺ T cells and CD4 ^- PBMC cells using an anti-CD4 antibody;
(c ') using an anti -CD25 antibody and wherein the antibody -CD127 PBMC- derived CD4 ⁺ T cells, CD4 ⁺ CD25 ^high CD127 ^- the steps of the fractionation and ^{CD4 + CD25 high Treg (regulatory T} cell);
(c ") irradiating the CD4 ^- PBMC of the step (b) with radiation;
(d) Stimulating CD4 ⁺ CD25 ^high CD127 ^- Treg of step (c ') by the following combination
The magnetic poles are CD4 ⁺ CD25 ^high CD127 ^- wherein 1 to a ratio of 20 range with respect to Treg in the (c ") stage CD4 ^- PBMC, the IL-2, 1 to 10 ng / ml range of 100 to 2,000 units / ml range TGF-ss, anti-CD3 antibodies, and anti-CD28 antibodies; and
(e) repeating step (d) at intervals of 5 to 10 days one to ten times,
Wherein said composition promotes in vivo proliferation of immunoregulatory CD8 ⁺ CD28 ^- T cells. delete 2. The composition according to claim 1, wherein the immunoregulatory T cells are autologous, allogeneic or heterologous. delete delete The composition for suppressing immune response according to claim 1, wherein the composition is used for cell, tissue or organ rejection. The method of claim 1, wherein the composition is used to inhibit or reverse the rejection of a tissue graft by a graft receptor, to prolong survival or to preserve the function of the tissue grafted to the graft receptor, Of the composition. delete delete 3. The composition of claim 1 wherein the immunosuppressant is selected from the group consisting of ATG (anti-thymoglobulin), sirolimus, methylprednisolone, corticosteroids, antiproliferative agents, cyclosporine, tacrolimus, mycophenolate mopetil, Wherein the composition is selected from the group consisting of algin, brachyunal sodium, leflunomide, azaspirane, and combinations thereof. Immunoregulatory T cells derived from primates except humans and regulated by T cells; Anti-CD154 monoclonal antibody which is a CD40-CD40L binding inhibitor; A method of inhibiting an immune response in a primate, excluding a human, comprising administering to the transplanted porcine pancreatic tissue an immunosuppressive composition comprising an immunosuppressive agent,
Said immunoregulatory T cells comprising (a) separating PBMC (peripheral blood mononuclear cells) from the blood of a primate;
(b) dividing the isolated PBMC into CD4 ⁺ T cells and CD4 ^- PBMC cells using an anti-CD4 antibody;
(c ') using an anti -CD25 antibody and wherein the antibody -CD127 PBMC- derived CD4 ⁺ T cells, CD4 ⁺ CD25 ^high CD127 ^- the steps of the fractionation and ^{CD4 + CD25 high Treg (regulatory T} cell);
(c ") irradiating the CD4 ^- PBMC of the step (b) with radiation;
(d) Stimulating CD4 ⁺ CD25 ^high CD127 ^- Treg of step (c ') by the following combination
The magnetic poles are CD4 ⁺ CD25 ^high CD127 ^- wherein 1 to a ratio of 20 range with respect to Treg in the (c ") stage CD4 ^- PBMC, the IL-2, 1 to 10 ng / ml range of 100 to 2,000 units / ml range TGF-ss, anti-CD3 antibodies, and anti-CD28 antibodies; and
(e) repeating step (d) at intervals of 5 to 10 days one to ten times,
Wherein the composition promotes in vivo proliferation of immunoregulatory CD8 ⁺ CD28 ^- T cells. delete delete

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