MXPA99010893A - Composition and method to prevent graft rejection and other counter-adaptive t lymphocyte mediated immune responses - Google Patents

Abstract

A method is provided for preventing and reversing acute allograft rejection wherein both the CD80/CD86:CD28/CTLA-4 interaction and the CD40:CD154 interaction are interrupted. The effect of the method on treating autoimmune diseases and allergy is also set forth.

Description

"COMPOSITION AND METHOD TO PREVENT - THE REJECTION OF GRAFT AND OTHER IMMUNE MEDIA ANSWERS WITH THE LYMPHOCYTE T CONTRA-ADAPTIVE " "" FIELD OF THE INVENTION This invention relates to the field of tissue transplantation, and more particularly to the use of monoclonal antibodies specific for T cell determinants by blocking the cell-mediated immune responses resulting in allograft rejection or xenograft rejection. This invention also relates to the prevention or inversion of rejection of the graft organ and other immune responses mediated with anti-adaptive T lymphocyte The invention provides compositions and an order and method of treatment to reduce, or prevent graft rejection. of organs in primates or man, and to prevent the disease resulting from a poorly focused T lymphocyte-mediated immune response.

BACKGROUND OF THE INVENTION The transplantation of organs between non-genetically identical people invariably results in the immunological rejection of the organ through mechanisms dependent on the T cell, unless the rejection process is repressed by administering drugs that suppress the function of the T cell. Calcineurin phosphatase inhibitors such as glucocorticosteroids are used clinically, and both prevent the release with T-cell activation of cytokines, particularly IL-2. Therapy with these agents is, however, important. Both act by deteriorating the signals through the T cell antigen receptor (TCR) and the sole mediator of the specificity of the T cell antigen that act on all T cells indiscriminately. In addition, the effect of these drugs is not lasting, so that the cessation of immunosuppression has generally resulted in graft loss even after survival free from prolonged rejection. In this way, in order to avoid rejection of the graft, transplant recipients must suffer the consequences of non-specific immunosuppression. These consequences include an increased risk of infection and malignancy, as well as significant cost and toxicity related to the drug. The data that establish that T-cell activation requires both TCR-mediated signals and cost-stimulating signals delivered simultaneously have accumulated over the last 20 years [1]. These important cost stimulatory signals are provided at least in part by the CD28 molecule based on the T cell when bound to its CD80 (B7-1) or CD86 (B7-2) counter-receptors, to which reference will be made. then jointly as B7, in cells presenting the antigen (APCs) and possibly parenchymal cells [1, 2, 3]. The interaction of CD40 and its coordinating group based on the T cell, CD40L (CD154), also plays an important role in the activation of the T cell, at least in part by regulating B7 in a way [4, 5]. In addition, CD40 and CD154 have a pivotal role in establishing T cell activity dependent on T [6, 7]. Also, studies have shown that the CTLA4 cell molecule (CD152) appears to regulate the cost stimulus and TCR-mediated activation, at least in part competing with CD28 and B7, and providing a negative signal. singular to the TCR signal transduction complex [8]. Several groups have shown in rodents that activation of the T cell can be blocked and the survival of the rodent allograft can be prolonged by interfering with B7 that interacts with its CD28 counter-receptors and T-cell CTLA4 using the B7-specific fusion protein, CTLA4-Ig [9-11]. Others have shown that the binding regulation of B7 can be prevented by the specific monoclonal antibody of CD154, MRl [4]. Since both agents seem to depend on the TCR coupling for their efficacy, the specificity of the T cell response can be exploited theoretically instead of depending on the suppression of the T pan call. In addition to in vitro efficacy, these agents have been shown to be dramatic in their effects in vivo rodents, allowing the acceptance of completely unmatched skin grafts, a result not obtainable with the immunosuppression available at present [12]. It should be noted, however, that all the previously disclosed techniques that allow long-term graft survival in rodents have failed in terms of work or have been associated with greater toxicity when tested on higher species in the phylogenetic tree.

COMPENDIUM OF THE INVENTION Accordingly, an object of this invention is a combination of drugs to prevent the rejection of transplanted cells, tissues or organs that are either an allogenic source or a xenogeneic source by administering agents that interfere with the cost-stimulating signals of the T cell. through CD28, when administered together with agents that interfere with the interaction of CD40: CD154. Another object is a method of treatment that reverses the rejection of an ongoing organ by administering agents that interfere with the stimulatory signals at cost of the T cell through CD28 when administered together with agents that interfere with the interaction of CD40: CD154. A third object recognizes that the reversal of an ongoing rejection process can be stopped by administering agents that interfere with the stimulatory signals at cost of the T cell through CD28 when administered together with agents that interfere with the interaction of CD40: CD154. A fourth object is that for patients who are currently being treated with normal immunosuppressive therapies (eg glucocorticoids, calcineurin phosphatase inhibitors, mycophenolate mofetil) to prevent rejection of a transplant or to prevent graft versus host disease, those medications toxic and expensive drugs could be discontinued and replaced with short-course therapy with agents that interfere with the stimulatory signals at cost of the T cell through CD28 when administered together with agents that interfere with the interaction of CD40: CD154. A fifth object is that for patients with a chronic rejection that undergoes the transplanted organ, the agents that interfere with the stimulatory signals in cost of the T cell through CD28 when administered together with agents that interfere with the interaction of CD40: CD154 , can block this unwanted immune reaction. A sixth and more general object is to prevent and / or treat disease states resulting from a counter-adaptive immune response such as autoimmune diseases mediated with T-lymphocyte (eg insulin-dependent diabetes mellitus, multiple sclerosis, etc.). and several states of allergic disease (eg hay fever). A seventh object is to test the hypothesis that CTLA4-Ig and anti-human CD154-specific monoclonal antibody are capable of inducing tolerance to tissues allografted or a xenografted in humans, and in a more general sense to alleviate (prevent or treat) all states of disease mediated with the anti-adaptive T lymphocyte. These and additional objects of the invention are achieved by: 1. Using agents that interfere with the interaction of CD28 and / or CD152 (CTLA4) and their coordinating groups of the B7 family (CD80 and / or CD86) and with interfering agents with the interaction of CD40 and CD154 (CD40L). These agents will be administered parenterally (intramuscularly, subcutaneously and especially preferably intravenously) in a normal pharmaceutical carrier (i.e. iv infusion with saline, water or other stabilizing agent). 2. The agents will be administered after the cells, tissue (s) or organ (s) have been transplanted. The initial dosage will be administered as soon as the graft is transplanted at a dose of between 5 to 20 milligrams per kilogram of body weight (each agent). The doses will then be administered on days 2, 4, 6, 8, 12, 16 and 28 of the post-transplant. Then, if they follow signs of immune rejection, the dosage will be repeated to reverse the rejection episode. During this retreatment, the dosage will be administered in accordance with the post-transplant of the initial induction therapy. 3. This therapy employing agents that interfere with the interaction of both CD28 / CD152: B7 and CD40: CD154 will also be administered to people with signs that they are developing a disease (including chronic rejection) or that they are already suffering from a disease, mediated completely or in part by activated T cells (including patients with a transplant who currently receive normal immunosuppressive therapy). These "anti-adaptive" T cell responses also include diseases similar to various autoimmune diseases (e.g., insulin-dependent diabetes mellitus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel or stomach disease, and systemic lupus erythematosus) as well as states resulting from the sequence of an immune response such as an allergic disease (hay fever). For these indications, the therapy will be administered in doses ranging from 2 to 20 milligrams per kilogram of body weight (each agent) as frequently as every other day for up to 28 days. 4. The "treatment package" will be referred to as "immune re-education" and will consist of drugs to be administered, the carrier solvent for those agents, and the infusion system to be used to administer the agent. This hypothesis is tested in a related preclinical model. CTLA4-Ig and anti-CD154 were tested alone in combination in rhesus peripheral blood leukocytes in vitro, and in rhesus monkeys transplanted with mainly vascularized renal allografts.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. The effect of CTLA4-Ig and humanized antihuman CD154 alone and in combination in unidirectional rhesus monkey mixed with lymphocyte reactions. Increasing CTLA4-Ig concentrations results in progressive suppression while the effects of humanized anti-human CD154 are more modest. The combination is more effective than any single drug at concentrations greater than 100 times. The results shown were produced in three independent experiments. C.P.M. = per minute counts of incorporated H-thymidine. Figure 2. (A) Survival and renal function, as determined by serum creatinine after unchanged allogeneic renal transplantation (hyphens) or transplantation after induction with CTLA4-Ig alone (squares) or humanized anti-human CD154 alone (diamonds). Open arrows indicate re-treatment during rejection demonstrated with biopsy. The solid arrows continued to survive. (B) Survival and renal function, as determined by serum creatinine after unmodified allogeneic renal transplantation (scripts) or transplantation after induction with both CTLA4-Ig and humanized anti-human CD-154. The open circles indicate the treatment on days 0, 2, 4, 6, 8, 10 and 12 of post-transplant. The closed circles indicate the treatment on days 0, 2, 4, 6, 8, 16 and 28 of post-transplant. Open arrows indicate retreatment during rejection demonstrated by biopsy for the animal illustrated in open circles. Solid arrows indicate continuous survival free of rejection from the transplant. Figure 3. (A) Histology of the renal allograft showing acute cellular rejection after unmodified renal allograft in rhesus monkeys. (B) Histology of the renal allograft showing the cell rejection -acute before inversion with humanized anti-human CVD154. (C) Normal renal allograft histology of an animal with normal renal function 163 days after transplantation and induction with CTLA4-Ig and CD154-humanized anti-human. (D) A perivascular lymphoid aggregate with the allograft shown in C. These lymphocyte nests exist in the allograft despite normal function and absence of immunosuppression. All micrographs are 250 times. Figure 4. Mixed lymphocyte responses against donor lymphocytes and lymphocytes of the third part for two rhesus monkeys, 150 days after the transplant with survival free of rejection and normal renal function and without any chronic therapy. The responses of both the donor and the third party are maintained. On the other hand, in the data NOT shown, the skin grafts placed in the rhesus monkey 6 months after the successful alotransplantation revealed specific tolerance of the donor. Three skin grafts were placed: one from the host (an autograft to control the surgical technique), one from the allogeneic kidney donor and one from a donor that is the third part. Only one donor skin of the third part was rejected during day 14 (and counting) from the graft, this data indicates that the specific tolerance of the functional donor has been achieved despite the failure of the allo-MLR to reflect it. A more complete appreciation of the invention will be readily obtained by reference to the following description of the preferred embodiments and the accompanying drawings wherein the like numbers in the different figures represent the same structures or elements. The representations in each of the figures is diagrammatic and no attempt has been made to indicate the real scales or the precise relationships. Proportional relationships are shown as approximations.

DESCRIPTION OF THE PREFERRED MODALITIES - This invention is applicable to both xeno-transplants and allo-transplants, and to a more general application to disease states resulting from lymphocyte responses. T counter-adaptive. The invention comprises a composition that involves parenteral administration for an agent that interferes with T-cell cost-stimulating receptors (CD28-CD152) the ability to bind to B7 in a tight time sequence for administration of an agent that prevents signals from through CD152. The best known mode so far of the initial experience in primates with a new class of reagents aimed at modifying the cost stimulus of the T cell, instead of focusing on the suppression or elimination of the T cell. Here, the strategies designed to interfere with the interaction of B7 and its CD28 and / or CD152 counter-receptors, or with the regulation of the B7 expression is shown as having dramatic effects on the T cell response in vitro, and on the survival of allograft in vivo - including the prevention of rejection and reversal of rejection demonstrated by established biopsy. In addition, these data demonstrate that the anti-rejection activity may persist long after the administration of the drug has stopped. Finally, the data is presented to indi that tolerance can be achieved to a specific donor. It is encouraging that this regimen was remarkably simple involving two agents administered through a normal peripheral intravenous probe and that it was also tolerated by means of the receptors. This is in marked contrast to other regimens used to achieve the acceptance of durable graft in primates that require ionization radiation, donor administration of bone marrow and significant perioperative immunosuppression [15,16]. The animals treated in this study did not present evidence of T cell activation or cytokine release typically observed after treatment with antibodies directed to CD3, and prolonged survival has not been carried out with it at a demonstrable cost in terms of opportunistic infection. In addition, alterations in peripheral blood haematological parameters were not observed during these studies. Long-term survival was achieved without apparent clearance or overall reductions in any subset of lymphocytes and no loss of T cell response in vitro. Therefore, it is unlikely that the observed effect is attributable to the destruction of the T cell after opsonization of antibody protein or fusion. The results are remarkable. This success is that rhesus monkeys suggest that allograft tolerance is a goal that can be achieved in humans using this approach or a similar therapeutic approach. The mechanism and the relative contribution of each agent remains as a matter of speculation during this meeting. Successes of CD154 blockade only suggest that any basal co-stimulus signal is less important to maintain the rejection response than the regulation of B7. Of course, anti-CD154 resulted in the survival free of impressive rejection when used alone, whereas the effects of CTLA4-Ig were not transient. Given the recent discovery that CD154 is expressed in non-myeloid cells such as vascular endothelium and smooth muscle [17], "and that B7-1 can be induced in fibroblasts [3] and hepatocytes [18], events that are not The T cell may be critical for establishing the reactivity against the allograft, denying access to the immune system for significant parenchymal adherence and cost-stimulating signals at the time of transplantation, graft recognition and destruction may be prevented. donor parenchyma and activation induced by lymphoid cells could explain the conservation of in vitro activity for donor lymphocytes despite the normal graft function, and the overall unsatisfactory correlation between MLR reactivity and the result of the clinical graft. However, the effects of CTLA4-Ig and anti-human CD154 managed showed that they were synergistic both in vitro as in vivo. Possibly, CTLA4-Ig provides security against expression of B7 that escapes the effects of humanized anti-human CD154. In that case, it seems that considerable time is required to mount an effective acute rejection with the few cells that escape the initial blockade. How this strategy was successful to reverse the acute rejection demonstrated by the established biopsy, it would seem that the rejection process must be maintained by continuous co-stimulation rather than a process that once set in motion advances unless the effector cells are eliminated or rendered incapable of TCR signals. Teleologically, the body is better served by inflammation which is easily controlled. Therefore, in the absence of attack direction, the re-treatment may be of unspoken order. This suggests that exploitation of the natural propensity of the immune system to regulate descendingly may be more advantageous than pan-suppression. The rhesus monkey model used in this study has repeatedly been shown to be a rigorous test of immune manipulation - one that is exquisitely sensitive to even small changes in allograft function or detrimental effects in the healing of the recipient's wound and immune function [13, 15, 19]. In addition, it has obvious biological similarity to human kidney transplantation. Specifically, the genes encoding MHC proteins are well known among rhesus monkeys and humans [20-22], and their rejection of closely parallel vascularized organs that appear clinically [13, 15, 19]. However, the issues of optimal dosing and treatment time course remain to be resolved. While the rodent models have been satisfactory with a single dose of CTLA4-Ig that is administered during the post-operative day 2 in combination with the specific transfusion of the donor [9], it is clear that a more aggressive approach is required in primates. However, a well-tolerated treatment "Transient that exploits the specificity of the immune system and provides survival free from lasting rejection would appear to be close to clinical applicability." Having described the invention, the following examples are provided to illustrate the specific applications of the invention, including the best so far known to carry out the invention. These specific examples are not intended to limit the scope of the invention described in this application.

- - MATERIALS AND METHODS Reagents Human CTLA4-Ig and a control fusion IgGl protein were prepared as described above [2] and embarked on a solution using the Genetics Institute of Cambrigde, MA. The humanized anti-human CD154 of the anti-CD40 coordinating group antibody was prepared as described previously [6, 7] and shipped in solution by Biogen Corporation, Cambridge, MA. The marionate anti-mouse CD28 monoclonal antibody PV-1 (IgG1, clone G62) was purified from the supernatants of the hybridoma culture and used as the isotype control monoclonal antibody.

MHC Type and Donor-Receptor Selection The donor-recipient combinations and selected animals for the third-party cells were selected based on the non-genetic identity of both MHC class I and class II. The disparity of class I was established by a one-dimensional isoelectric approach as described previously [13]. The disparity of class II was established based on the - results of unidirectional mixed lymphocyte reactions (MLRs). In addition, the animal's DRB sites were verified as being unequal by denaturing gradient gel electrophoresis and direct sequencing of the second DRB exon, as described previously [14]. The response of the vigorous in vitro T cell of the recipient to the donor was confirmed in vitro for all donor-recipient pairs. The experiments described in this study were carried out in accordance with the principles outlined in the "Guide for the Care and Use of Laboratory Animáis," Institute of Laboratory Animáis Resources, National Research Council, DHHS, Pub. No. (NIH) 86 -23 (19850).

Cellular Analysis In Vitro Unidirectional MLRs were carried out on all animals _ before transplantation and on survivors exempt from rejection after 100 days. Each animal was tested against all potential donors to establish the highest responding pairs for transplantation. The response cells (3 x 10) were incubated with irradiated stimulant cells (1 x 10) at 37 ° C for 5 days. Cells were pulse labeled with 3 H-thymidine and proliferation was monitored by 3 H-thymidine incorporation. The polyclonal stimulus with - Concanavilin A (25 mcg / milliliter) served as a positive control. A stimulus index was calculated by normalization for self-reactivity, which in all cases was about background incorporation. For in vitro dose response studies, humanized anti-human CTLA4-Ig or CD154 was added to the MLR on day 1 at concentrations ranging from 100 mcg / milliliter to 0.01 mcg / milliliter. Combined treatments were carried out by varying the concentration of CTLA4-Ig and retaining the concentration of constant humanized anti-human CD154 at 50 mcg / milliliter. Peripheral blood lymphocyte phenotype analysis was carried out before transplantation and periodically during and after drug therapy. Trials were evaluated at 0.2 milliliter of heparinized whole blood diluted with phosphate-stabilized saline and 1 percent fetal calf serum. The FITC labeled Til, Bl (Coulter), and FN18 (the generous gift of Dr. David M. Neville, Jr.) monoclonal antibodies were used to assess the percentage of CD2 (T cell / cell NK), CD20 (cell B), and CD3 (T cell) positive cells respectively. The red cells were removed from the preparation by an ACK lysis stabilizer (0.15 M NH4CI, 1.0 mM KHCO3, 0.1 mM a2 EDTA, pH 7.3) treatment after dyeing. The cells were immediately subjected to flow cytometry or after fixation in 1 percent paraformaldehyde. Flow cytometry was carried out using Becton Dickinson FACSCAN.

Kidney allografts Renal allograft was performed as described previously [13]. Briefly, juvenile rhesus monkey (1 to 3 years old), seronegative for simian immunodeficiency virus, simian retroviruses, and herpes B virus, were obtained from the Primate Center (University of Wisconsin) or LABS (Yemassee, SC ). The procedures were carried out under general anesthesia using ketamine (1 milligram per kilogram, i.m.), xylazine (1 milligram per kilogram, i.m.) and halothane (1 percent, inhaled). The transplantation was carried out between genetically distinct donor-recipient pairs as described by the MHC analysis described above. Animals were heparinized during organ harvesting and implantation (100 units per kilogram). The allograft was implanted using normal microvascular techniques to create end-to-side anastamosis between the donor renal artery and the distant aorta of the recipient as well as the renal vein of the donor and the vena cava of the recipient. Then one was created - primary ureteroneocystostomy. The bilateral native nephrectomy was completed before closure. The animals were treated with intravenous fluid for approximately 36 hours until oral administration was adequate. Trimetaprim-sulfa was administered for 3 days after surgical antibiotic prophylaxis. Each animal received 81 milligrams of aspirin on the day of surgery. The need for analgesia was evaluated frequently and the analgesia was maintained with intramuscular butorphenol. The animals were weighed weekly. The sutures of the skin were removed after 7 to 10 days. Humanized anti-human CTLA4-Ig and / or CD154 intravenously was intravenously administered intravenously at dosing and dosing schedule which varied based on the accumulated experience with the agents. No immunopharmaceuticals were administered. Serum creatinine and whole blood electrolyte (Na *, K *, Ca2 *) and hemoglobin were determined every other day until stable and then weekly.

Pathological Analysis Biopsies were performed on animals suspected of rejection using a 20-gauge needle-core device (Biopty-Cur, Bard). Normal staining with hematoxylin and eosin was carried out - in frozen tissue or fixed with formalin to confirm the diagnosis of rejection. The animals were euthanized at the same time from the anuria or if a weight loss of 15 percent of the body weight of the pretransplant had occurred in accordance with AAALAC standards. All animals underwent complete gross and histopathological evaluation at the time of death.

RESULTS CTLA4-Ig and humanized anti-human CD154 that synergistically prevent the proliferation of the T cell in vitro. Both CTLA4-Ig and humanized anti-human CD154 inhibited the rhesus MLRs in a dose-dependent manner (Figure 1). CTLA4-Ig, however, was an effective humanized anti-human CD154 as a single agent to prevent T cell proliferation. Considerable reduction in thymidine incorporation was seen at a CTLA4-Ig concentration of 0.1. mcg / milliliter, and additional inhibition was achieved at higher concentrations. The modest reduction in proliferation was achieved with humanized anti-human CD154 concentrations of 0.01 mcg / milliliter but the inhibition did not improve considerably increasing the concentrations. Both agents acted synergistically, inhibiting the proliferation combination by approximately 100 times more effectively than any agent alone did. The dose response studies were repeated for 3 separate animals with identical results. CTLA4-Ig and humanized anti-human CD154 synergistically prevent allograft rejection in vivo. Twelve renal allograft transplants were carried out (Figure 2). Four animals received the transplants without any immunological intervention. These animals rejected on days 5, 7, 7 and 8. The histological examination of their kidneys shows acute cellular rejection characterized by diffuse interstitial and tubular lymphocytic infiltration with edema and cell necrosis (Figure 3A). An animal was administered in a course of 5 days CTLA4-Ig (10 milligrams / kilogram / day) beginning at the time of transplantation and had prolonged graft survival at 20 days (Figure 2A). The graft loss was due to cellular rejection not distinguishable from that seen in the control animals. One animal was treated with CTLA4-Ig 20 milligrams per kilogram on the day of transplantation followed by a 12-day course of 10 milligrams per kilogram every other day and had prolonged graft survival up to 30 days (Figure 2A). Again, graft loss was due to acute cellular rejection. Extrapolating from the work previously published in the heterotopic cardiac allograft model of Turka, and others [9] a specific transfusion of the lymphocyte-derived lymphocyte donor was administered (10 *) at the time of transplantation to these 2 animals. Two animals were treated with humanized anti-human CD154 alone (Figure 2A). Both animals received 20 milligrams per kilogram every other day beginning on the day of surgery and continuing for 14 days after the operation (in total 8 doses). Both animals experienced survival free from prolonged rejection even when transient creatinine elevations were recorded during the second and fourth weeks of post-operation. Both animals were rejected between 95 and 100 days post-transplant. The biopsy was carried out on each animal to confirm the diagnosis (Figure 3B). Both animals were then treated again with 7 doses of the humanized anti-human CD154 (20 milligrams per kilogram, one animal every other day and one animal daily) and both returned to the normal graft function without demonstrable detrimental effects. They remain alive and in good condition more than 150 days after the transplant at the time of this writing. Two animals were administered 20 milligrams per kilogram each of CTLA4-Ig and the humanized anti-human CD154 after transplantation (Figure 2B). Again, each drug was administered every other day beginning on the day of surgery and continuing for 14 days after the operation. One animal was rejected 32 days after surgery. The others remained free of rejection after 100 days, but just as those animals treated with humanized anti-human CD154 were only rejected during that time. Similarly, a biopsy showed acute cellular rejection. The initial regimen of CTLA4-Ig and humanized anti-human CD154 was repeated and the creatinine returned to the baseline (1.0). The analysis of MLR after this treatment showed a loss of specific reactivity of the donor. The answer of the third part remained. At 165 days post-transplant, the animal was sacrificed as required by the protocol due to weight loss. The graft function during that time was normal. During the autopsy, the animal was found to have shigella and canfilobacter enterocolitis, a common infection in rhesus monkeys. This disease had infected multiple animals in the original primate colony, including several untreated animals. No other pathological abnormality was found; specifically, there was no evidence of lymphoproliferative disease or opportunistic infection. Histologically, the graft had isolated nests of lymphocytes in the interstitium, but no evidence of tubular infiltration, glomerular damage, or parenchymal necrosis (Figure 3C). Like animals treated with humanized anti-human CD154 alone, both of these animals had transient increases in their creatinine combined with an increase in graft size during the fourth postoperative week. It was hypothesized that this graft swelling reflected a second wave of infiltrating lymphocytes and thus led to a modified dosage schedule such that both reagents were administered on the day of surgery and in the post-operative days. 2, 4, 6, 8, 12, 16 and 28. Two animals were treated with this modified regimen (Figure 2B). Both had experienced survival free of rejection, exemption from disease or alterations in renal function for more than 150 days. Both remained alive and well at the time of this writing. After 100 days of rejection-free survival, the MLRs were repeated against the donor cells and the third-party cells. No changes in in vitro reactivity were observed (data not shown). These studies were repeated after 150 days of rejection-free survival with identical results (Figure 4). Both animals maintained vigorous responses in vitro to the donor and the third-party cells but stopped rejecting their allografts. No animal showed toxicity of any of the therapies employed. Specifically, there was no fever, anorexia, or hemodynamic abnormalities, and opportunistic infections had not occurred. The animals have moved away under normal conditions and have been allowed to contact other animals in the colony. They have maintained normal weight gain. Laboratory chemistries and hematological parameters such as hemoglobin and leukocyte counts have remained normal. The percentages of cells expressing CD2, CD3 and CD20 were not affected by any treatment regimen (data not shown). Specifically, no reductions in T cell counts were observed during or after treatment in any animal. Obviously, many modifications and variations of the present invention are possible in view of the aforementioned teachings. Therefore, it should be understood that, within the scope of the appended claims, the invention may be put into practice in a manner other than that specifically described.

Claims (16)

1. A composition for the suppression of agents that compromise the rejection of transplantation of organs that disrupt the interaction of B-7 (CD80 / CD86): CD28 / CD152 in combination with agents that interfere with the interaction of CD40: CD154.

The composition of claim 1, wherein the B-7: CD28 switch is selected from the group consisting of CTLA4-Ig, anti-CD80 antibody, anti-CD86 antibody, anti-CD28 anti-pro-drug, anti-CD152 antibody and fragments and modifications of that switch and the CD40 switch: CD154 and fractions and modifications of that switch, an anti CD154 antibody.

3. The composition of claim 2 comprising from about 5 to 20 milligrams per kilogram of switch-B-7: CD28 and from 5 to 20 milligrams per kilogram of switch CD40: CD154.

The composition of claim 1 wherein the agents interfere with the interaction of CD28 and / or CD152 (CTLA4) with their coordinating groups of the B7 family (CD80 and / or CD86) and with agents that interfere with the interaction of CD40 and CD154 (CD40L).

5. A treatment regimen for suppressing rejection of organ transplantation and inducing tolerance comprising administering at least 2 doses of a B-7 switch agent: CD28 in combination with a CD40: CD154 breaker agent at least in the first 5 days after the transplant resposition of an organ.

6. The treatment regimen of claim 4, wherein from 5 to 20 milligrams per kilogram body weight of each switch agent combination B-7: CD28 with the CD40: CD154 switch is administered until every 2 to 4 days during the first month after the transplant. The treatment regimen of claim 5 wherein the combination of agents is administered together. 8. The treatment regimen of claim 5, wherein the combination of agents is administered separately on the same day. - Claim 9. The treatment regimen of claim 6, wherein the switch of B-7: CD28 is selected from the group consisting of CTLA4-Ig, the anti-CD80 antibody, the anti-CD86 antibody, the anti-antibody. CD28, anti-CD152 antibody and fragments and modifications of that switch and switch "of CD40: CD154 and fractions and - modifications of that switch, and the anti-CD154 antibody. The treatment of claim 6, wherein the combination is administered parenterally. The treatment regimen of claim 6, wherein the combination is administered by means which are selected from the group consisting intramuscularly, subcutaneously and intravenously in a normal pharmaceutical carrier. 12. The treatment regimen of claim 5, wherein the treatment is administered to reverse the inverse reverse organ rejection by administering agents that interfere with the cost-stimulating signals of the T cell through CD28 when provided together with agents that interfere with the interaction of CD40: CD154. The treatment regimen of claim 5 wherein the treatment is administered to patients receiving immunosuppressive drugs to wean them from the immunosuppressive drugs. 14. A treatment of disease state resulting from a counter-adaptive immune response in such a manner that the different T-lymphocyte-mediated autoimmune diseases (e.g., diabetes mellitus depending on insulin, multiple sclerosis, etc.) and various states of - allergic disease (e.g., hay fever) comprising administering at least 2 doses of a B-7 switch agent: CD28 in combination with a CD40: CD154 switch agent in at least 5 days. The treatment of claim 5 wherein the initial dosage is administered as soon as the graft is transplanted at a dose of between 5 to 20 milligrams per kilogram of body weight (of each agent) and the doses are administered on days 2, 4, 6, 8, 12, 16 and 28 of the post-transplant. 16. The treatment of claim 15 wherein the dosage will be repeated to reverse the rejection episode if it continues in an immune rejection, and during this re-treatment, the dosage is administered in accordance with post-transplant therapy. initial induction.