JP2002541195A - A composition comprising an immunotoxin and an agent that inhibits maturation of dendritic cells for inducing immune tolerance to a graft - Google Patents

Abstract

The present invention provides a method for inducing immune tolerance to a transplant in a recipient, the method comprising administering an immunotoxin to the recipient, thereby reducing the recipient's T cell population. Allowing the agent to inhibit the maturation of dendritic cells is then administered to the recipient. The present invention also provides a method for screening an agent that acts synergistically with an immunotoxin in inducing immune tolerance and a method for screening an agent that inhibits maturation of dendritic cells. The invention also provides a method of treating a subject having an autoimmune disease, the method comprising administering an immunotoxin to the subject, thereby reducing the T cell population of the subject; An agent that inhibits maturation is administered to a subject. Also provided is a composition comprising an immunotoxin and an agent that inhibits dendritic cell maturation.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a method of inducing tolerance using an immunotoxin in combination with an agent that inhibits dendritic cell maturation. The present invention is in the field of immunobiology.

[0002] Technical Background graft tolerance, eliminating the need to maintain the non-specific immunosuppressive agents uncertain, the patient and the ideal to make successful xenotransplantation is without side effects associated with their It is a goal for doctors. As with many transplant procedures, it is difficult to secure live allografts. In addition, long-term immunosuppression can cause bile in the phylum.

[0003] Over the past decade, most transplant recipients have been treated with cyclosporine, azathioprine, and various other immunosuppressive drugs to maintain immunosuppression. Average annual cost of maintaining immunosuppressive treatment in the United States is around 1000
$ 0. In addition to cost, these agents have considerable side effects due to their non-specific effects, which include impaired cell and organ function, increased susceptibility to infection. The primary goal of transplant immunology is to generate specific immunological tolerance to the graft while relieving the patient of the side effects of continuous pharmacological immunosuppression and the complications and costs associated therewith.

[0004] Anti-T cell therapy (anti-lymphocyte globulin) has been used in rodents with thymic injection of donor cells (Posselt et al. Science 1990; 249: 1293-1).
295 and Remuzzi et al. Lancet 1991; 337: 750-752). Although thymic tolerance has proven successful in rodent models, it requires exposing the recipient's thymus to an alloantigen of the same donor before transplanting an organ allograft from the donor. However, thymic tolerance has not been reported in large animals, and its relevance to tolerance in humans is unknown.

[0005] One approach to achieving immune tolerance has been to expose recipients to cells from donors prior to transplantation in hopes of inducing tolerance to subsequent transplants. This approach involved placing donor cells (eg, bone marrow) presenting MHC class I antigens immediately after anti-lymphocyte serum (ALS) or irradiation in the recipient's thymus. However, this approach has proven difficult to apply to living primates (eg monkeys or humans). AL
S and / or irradiation renders the host susceptible to disease or side effects and is ineffective.

[0006] If a reliable and safe approach for specific immunological tolerance to transplantation, especially xenograft transplantation, is available, such an approach would be of enormous value and would have a world-class patient and transplant It will appeal to physicians, be immediately applicable to new transplants, and potentially provide a stable graft function to the graft present in the recipient. Thus, very specific induction of immune tolerance is desired. Furthermore, there is a need for a means for conferring tolerance in primates without the adverse effects of using ALS or radiation. Moreover, the goal is to achieve tolerance, not just delay rejection. An important goal is to suppress rejection until it is no longer a factor that reduces the life expectancy of transplant recipients.

SUMMARY OF THE INVENTION The present invention provides a method for inducing immune tolerance to a transplant in a recipient, the method comprising administering to the recipient an immunotoxin, thereby reducing the recipient's T cell population. Allowing the agent to inhibit the maturation of dendritic cells is then administered to the recipient. The agent that inhibits dendritic cell maturation is administered to the recipient at least once, preferably on the day of transplantation. More preferably, an agent that inhibits maturation of dendritic cells within one to two weeks after transplantation is further added with four to one.
Administer 4 times to the recipient. If desired, an agent that inhibits dendritic cell maturation can be administered to the recipient prior to transplantation and / or can be administered to the donor prior to removing the graft. The agent that inhibits dendritic cell maturation is NfκB
And may include, for example, deoxyspergualin, methyl-deoxyspergualin, and other deoxyspergualin derivatives or analogs. Other analogs that inhibit dendritic cell maturation include, for example, soluble interleukin 17 (IL-17) receptor Fc fusion protein,
It may include glucocorticoids, blockers of tumor necrosis factor alpha binding, blockers of granulocyte macrophage colony stimulating factor binding, blockers of IL-12p70 binding, or blockers of IL-1β binding. Agents that inhibit dendritic cell maturation may also be blockers of immature dendritic cell epitopes or blockers of dendritic cell precursor cell epitopes involved in dendritic cell maturation, such as anti-CD40 ligand (ie, anti-CD154). And more particularly, the anti-CD40 ligand may be 5C8 or a derivative or analog thereof.

[0008] The present invention also provides a method of screening for an agent that acts synergistically with an immunotoxin in inducing immune tolerance, the method comprising: transplanting a donor graft into a recipient; Administering, thereby reducing the T cell population of the recipient; administering the agent to be screened to the recipient; obtaining a sample containing dendritic cells; then expressing a marker specific for mature dendritic cells Determining the percentage of dendritic cells in the sample that can be induced to express or express, wherein a lower percentage indicates synergy. A method for screening for an agent that inhibits maturation of dendritic cells is also provided by the present invention, the method comprising:
Obtaining a population of immature dendritic cells from a sample containing dendritic cells of interest; culturing the cell population in the presence of the agent to be screened; and then expressing or expressing a marker specific for mature dendritic cells Thus, the percentage of dendritic cells that can be induced is determined, with a lower percentage indicating inhibition of dendritic cell maturation. The invention also provides a method of treating a subject having an autoimmune disease, the method comprising administering an immunotoxin to the subject, thereby reducing the T cell population of the subject; An agent that inhibits maturation is administered to a subject.

[0009] Also provided is a composition comprising an immunotoxin and an agent that inhibits maturation of dendritic cells.
More specifically, the present invention provides that the immunotoxin is an anti-T cell immunotoxin directed to the CD3 epitope, or the agent that inhibits dendritic cell maturation is an inhibitor of nuclear translocation of NfκB. A composition is provided.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows induction of kidney graft tolerance by treatment of graft recipients with immunotoxin on days 0 and 1 in combination with DSG on days 0-14. Yes, significant Th2 cytokine polarization occurs. By week 2 post-transplantation, IL4 polarization (lower levels of IL4 compared to the group treated with immunotoxin alone and lower levels of IL4 compared to IFN-γ levels) in the group receiving the combination treatment Is shown. The group receiving DSG over 2 weeks showed a sustained and progressive increase in IL4 polarization at 4 weeks, whereas in the group receiving DSG for only 5 days, the polarization was no longer maintained and the cytokine pattern Was reversed. IL4 independent of the type of anti-CD3 immunotoxin, ie, total IgG or F (Ab) ₂ form
Polarization has occurred. FIGS. 2A, 2B, and 2C show the total IgG immunotoxin (FN18-CRM9), F
(Ab) RT-PCR performed on fresh rhesus monkey peripheral blood lymphocytes treated with either _two immunotoxins (FN18-F (Ab) ₂ -CRM9) or control buffered saline (PBS). The results are shown. FIG. 2A shows mRNAs for IL-2, IL-4, IL-10, and IFN-γ in the absence of DSG. FIG.
B: IL-2, IL-4, in cells treated with DSG (2.5 μg / ml)
2 shows mRNAs for IL-10 and IFN-γ. FIG. 2C shows the density ratio of various mRNAs to the actin control.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a short course of use of an immunotoxin that, when combined with an agent that inhibits dendritic cell maturation, prevents graft rejection while maintaining graft function. To provide a tolerance-inducing treatment rule. Thus, the present invention provides a method of inducing immune tolerance to a transplant in a recipient, the method comprising administering an immunotoxin to the recipient, thereby reducing the T cell population of the recipient; An agent that inhibits maturation of dendritic cells is administered to a recipient.

[0012] As used herein, "graft" may include a graft of allogeneic or heterologous organs, tissues, or cells. For example, grafts can be kidney, liver, heart, pancreas, lung, skin,
And pancreatic islets, hepatocytes, stem cell progenitor cells, and isolated cell transplants of differentiated stem cell progenitor cells.

As used herein, “recipient” or “subject” is preferably a mammal. More preferably, the mammalian recipient is a primate, even more preferably a human. The "recipient" or "subject" to be treated can be an individual human,
It can include domestic animals, livestock (eg, cows, horses, pigs, etc.), and pets (eg, cats and dogs).

A “donor” as used herein may be a dead donor or a living donor. Still further, the donor may be of the same species as the subject to be treated, or may be of a different species than the subject to be treated. Thus, transplantation between primate species (ie, xenograft or xenograft) using the methods of the present invention
As well as transplants (ie, allografts or allografts) in the same primate lineage. Even more sensitive xenografts can maintain function in the presence of this tolerance-inducing rule.

[0015] The donor graft used in the method of the present invention may contain cells that have been altered, for example, by genetically processing the donor or donor cells. For example, donor cells can be treated to reduce antigenicity or reduce the susceptibility of cells to immune injury (R. weiss, Nature 391: 327-28 (1998)).

As used herein, the term “agent for inhibiting maturation of dendritic cells” refers to that, when contacted with a dendritic cell precursor cell, all or a part of the precursor cell expresses a marker of a mature dendritic cell. Refers to an agent that inhibits Thus, an agent that inhibits maturation of dendritic cells will be more likely than it would be in the absence of membrane CD83, DR, or CD86 or nuclear Rel.
Cells expressing a marker such as -B at about 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
Reduce by 95 or 100%. Thus, as more fully described in the Examples herein, the efficacy of inhibitors on dendritic cell maturation is assessed by determining the percent reduction in the number of cells expressing a marker for mature dendritic cells. can do.

The agent that inhibits dendritic cell maturation may be an inhibitor of nuclear translocation of NfκB. More specifically, the inhibitor of nuclear translocation of NfκB may be deoxyspergualin or a derivative or analog thereof,
For example, include methyl-deoxyspergualin or a deoxyspergualin analog lacking a chiral center (e.g., LF08-0299) (Andoins et al., 1996, incorporated herein by reference). Other derivatives or analogs of deoxyspergualin may be used, for example, US Pat. No. 4,518,532.
No. 4,518,532, U.S. Pat. No. 4,525,299, U.S. Pat.
957504, U.S. Pat. No. 5,162,581, U.S. Pat. No. 5,476,870,
U.S. Pat. No. 5,637,613, WO 96/24579, EP600762, EP
669316, EP7433000, EP765866, and EP75538
0 (they are incorporated herein by reference).

[0018] Preferably, the agent that inhibits dendritic cell maturation comprises one or more NF-
Activate an AT-dependent Th2 cytokine (eg, one or more cytokines selected from the group consisting of IL2, IL4, and IL10). Also, the agent that inhibits maturation of dendritic cells is preferably one or more Nfκ
Inhibits B-dependent Th1 cytokines (eg, IFNγ). Such activation or inhibition may be due to an increase or decrease in cytokine gene expression, respectively. Thus, treatment with an agent can cause a change in the level of mRNA or protein levels encoding the cytokine. In one embodiment of the invention, the agent activates one or more NF-AT dependent Th2 cytokines and inhibits one or more NfκB dependent Th1 cytokines. Thus, for example, an agent activates IL4 and inhibits INFγ.

[0019] Alternatively, the agent that inhibits dendritic cell maturation is soluble IL-17 receptor Fc
Fusion protein, glucocorticoid, tumor necrosis factor alpha (TNF-α) binding blocker, granulocyte macrophage colony stimulating factor (GM-CSF) binding blocker, IL-12p70 binding blocker, or IL-1β binding blocker. You may. Agents that inhibit dendritic cell maturation also include blockers of immature dendritic cell epitopes or blockers of dendritic cell precursor cell epitopes involved in dendritic cell maturation, such as anti-CD40 ligand (ie, anti-CD154). More specifically, the anti-CD40 ligand may be 5C8 or an analog or derivative thereof. One skilled in the art will recognize that agents that inhibit dendritic cell maturation can be used in combination for either a synergistic or additive effect.

Preferably, at least one agent that inhibits dendritic cell maturation is added to the recipient.
Dosing once. More preferably, at least one day on the day of transplantation and the first week after transplantation.
1, 2, 3, on days 2, 3, 4, 5, 6, or 7 or the first two weeks after transplantation
Administer on days 4, 4, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Activation of cytokines mediated by the NF-AT transcription pathway and / or N
The route of administration may be optimized to sustainably promote inhibition of the fκB transcription pathway.
Thus, in one embodiment, the agent is administered daily on the day of implantation and 13 or 14 times thereafter.

In addition to administration on the day of transplantation and post-transplantation, an agent that inhibits dendritic cell maturation can also be administered to the recipient before transplantation. For example, 24 to 0.25 before transplantation
Hours ago, preferably 23, 22, 21, 20, 19, 18, 17, 16, 15,
14, 31, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5
An hour earlier, deoxyspergualin can be administered to the recipient. Furthermore, the present invention may be characterized in that an agent that inhibits dendritic cell maturation is administered to a graft donor at least once before removing the graft. For example, 24 to 0.25 hours before taking the graft, preferably 23, 22, 21, 20, 19, 18
, 17, 16, 15, 14, 31, 12, 11, 10, 9, 8, 7, 6, 5, 4
Deoxyspergualin can be administered to the donor at least once 3, 2, 1, 0.5 hours before. The route of administration of inhibitors of dendritic cell maturation prior to transplantation is limited by organ availability and unpredictable procurement, but the inhibitor may be administered to the recipient prior to transplantation and / or prior to obtaining the graft. Is preferably administered to the donor.

The preferred dose of the inhibitor of dendritic cell maturation will vary depending on the particular use inhibitor, the species, age, weight and general symptoms of the recipient, mode of administration, and the like. Thus, it is not possible or necessary to specify an exact dose, as an appropriate time course can be determined without undue experimentation by those skilled in the art. For example, the dose of deoxystergualine is 0.1 to 10 mg / kg / day,
. 5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5,
. 0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 1,
0.0 mg / kg / day or any amount in between.

As used herein, an “immunotoxin” may be an anti-T cell immunotoxin directed to the CD3 epitope. The immunotoxin may be an immunoconjugate of the fusion protein. The immunotoxin may be monovalent or divalent. Bivalent anti-T cell immunotoxin is UC
It may be HT1-CRM9 or a derivative or analog thereof. Bivalent anti-
The T cell immunotoxin may include a toxin moiety and a moiety targeting a T cell CD3ε epitope, and the toxin moiety may be a diphtheria toxin. The bivalent anti-T cell immunotoxin may be a processed bivalent fusion immunotoxin. Various immunotoxins and treatment rules are described in US application Ser. No. 09 / 06,413 and PCT / US98 / 0.
4303, which are incorporated herein by reference. The immunotoxin may be, for example, a whole IgG immunotoxin (FN18-CRM9), or the F (Ab) ₂ form of the immunotoxin (FN18-F (Ab) ₂ C
RM9).

In the present invention, the immunotoxin is administered at least twice, preferably at least on the day of transplantation and on the second day of transplantation. The immunotoxin can be administered 72 to 0 hours before and several days after transplantation. Preferably, the immunotoxin is a recipient,
Donors or both 72 to 48 hours before xenotransplantation and 2
It can be administered 4 to 0 hours before. Preferably, 1, 2 or 3 after implantation
The immunotoxin is administered to the recipient on or during the day. 4,5
Although administration of the immunotoxin can be used on days 6, 6 or 7, such an extended time course of immunotoxin administration requires that one begin with the production of anti-toxin antibodies beginning about five days after administration. There will be. The immunotoxin can be administered to the subject any time after the transplant. Thus, subjects with long-lived grafts that had not previously received immunotoxin treatment would still benefit from immunotoxin administration, thereby avoiding the need for long-term treatment with immunosuppressants. It will be. Graft recipients that have begun to show signs of rejection may benefit from immunotoxin administration in reducing or eliminating the rejection process. Thus, even in a recipient who has been previously treated with an immunotoxin, a recipient who shows a rejection response can be treated with a further administration of the immunotoxin.

In the present invention, preferably, the immunotoxin is one that temporarily reduces T cells in the blood and lymph nodes of a control or recipient by at least one log unit. Preferably, the number of T cells in the blood and lymph nodes is primarily 0.5.
7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
It will be reduced by 1.7, 1.8, 1.9, 2 or 3 log units, or by any value between 0.7 and 3 log units. "Primary depletion" is defined as a T cell loss of 0.1% in the blood and lymph compartment for at least 4 days.
It means decreasing by 7 to 3 log units and then beginning to return to normal levels.

The method of the present invention can be further characterized by administering an immunosuppressive agent to the recipient. As used herein, immunosuppressive agents include, for example, methylprednisolone, neoral, cyclosporine, mycophenolate moephytil, tacrolimus, rapamycin, steroids, or any combination thereof. The administration of immunosuppression can be started 24 to 0 hours before the transplantation, and the administration can be continued up to 2 weeks thereafter. Preferably, the immunosuppressant is at least 4,5,
It can be administered for 6, 7, 8, 9, 10, 11, 12, 13, 14 days or for a period in between. The combined dosing regimen of immunotoxins and dendritic cell maturation inhibitors allows for short-term immunosuppressant treatment and eliminates the need for long-term immunosuppressant treatment, thereby improving chronic immunity. The side effects associated with the inhibitors are avoided. Thus, when a course with an immunosuppressant is used in conjunction with an immunotoxin and an agent that inhibits maturation of dendritic cells, the course with the immunosuppressant may be shorter and / or may prevent graft rejection. The dose may be lower than conventionally used.

[0027] Additional therapeutic agents may be used with the methods of the invention, including inducing immune tolerance and targeting immune disorders. Thus, the present invention provides at least one method of inducing tolerance using an immunotoxin (IT), wherein the induction of tolerance comprises: (1) combining IT with an agent that inhibits dendritic cell maturation. Induction of tolerance by administration; (2) induction of tolerance by administration of IT, an agent that inhibits maturation of dendritic cells, and at least one or a combination of immunosuppressants. The additional therapeutic agent can be administered before, simultaneously with, or after administration of the immunotoxin. As described further below, different additional therapeutic agents can be administered to the recipient at different times or simultaneously with respect to the transplantation event or administration of the immunotoxin.

Since the immunosuppressant can be administered prior to treatment with the immunotoxin and the agent that inhibits maturation of dendritic cells, the present invention combines the immunotoxin with an agent that inhibits maturation of dendritic cells. The methods of the invention can be used with transplant recipients during a course of treatment with an immunosuppressive agent. This provides an important opportunity to reduce or eliminate conventional immunosuppressant treatment and its well-documented negative side effects. Further, an agent that inhibits maturation of dendritic cells before transplantation and / or
Alternatively, treatment with immunosuppressants can be particularly useful in post-mortem and xenografts. In the context of such pre-transplant therapy, administration of the immunotoxin can be delayed up to 7 days after transplant or later.

Examples of administration schedules for immunotoxins and agents that inhibit dendritic cell maturation for patients undergoing organ transplantation are as follows: Day 24-0 hours: Treat recipient with inhibitory agent (optional) Day 0: Transplant, administer first immunotoxin immediately after transplantation, treat recipient with agent that inhibits dendritic cell maturation Day 1: Treating the recipient with an agent that inhibits dendritic cell maturation Day 2: Administer a second immunotoxin and treat the recipient with an agent that inhibits dendritic cell maturation 3 Day: treating the recipient with an agent that inhibits dendritic cell maturation Day 6: treating the recipient with an agent that inhibits dendritic cell maturation; or-Day 24-0 hour: Tree Agents that inhibit dendritic cell maturation Treat the ent (if desired) Day 0: Perform the transplant, administer the first immunotoxin immediately after transplant, and treat the recipient with an agent that inhibits dendritic cell maturation Day 2: Second Day 4: Treating the recipient with an agent that inhibits dendritic cell maturation Day 7: Treating the recipient with an agent that inhibits dendritic cell maturation Day 10: Tree Treating Recipient with Agent that Inhibits Dendritic Cell Maturation Day 13: Treat the recipient with an agent that inhibits dendritic cell maturation.

In any case, the donor can be treated with an agent that inhibits dendritic cell maturation 24 hours to 0 hours before taking the graft, and / or before transplantation.
The recipient can be treated with an immunosuppressant on or after the transplant. Those skilled in the art will know to vary the time course depending on the individual recipient, depending on the species, age, weight and general symptoms of the recipient, the particular agent used, the mode of administration, etc. There will be. Thus, it is not possible or necessary to specify an exact time course. However, one skilled in the art can determine an appropriate time course.

A currently preferred dose of immunotoxin is a peripheral blood T cell level of 80% of the pre-injection level.
, Preferably a dose sufficient to deplete to 90% (or particularly preferably 95% or more). This would require mg / kg levels for humans as well as monkeys (eg, 0.05 mg / kg body weight to 0.3 mg / kg body weight).
kg body weight), studies of its toxicity have shown that it is more well tolerated by humans. Thus, an immunotoxin can be administered to safely reduce the recipient's T cell population.

The effectiveness of a method for inducing tolerance can be assessed using methods well known in the art. Graft function and clinical signs of graft rejection can be assessed. For example, when using a method to induce tolerance to pancreatic cell grafts or pancreatic islet cell grafts, blood glucose levels can be measured,
Preferably, non-fasting blood glucose levels should be maintained at less than 160 mg / dl upon completion of the tolerance induction regimen, indicating the absence of graft rejection. Other signs of graft rejection include intestinal fibrosis, intestinal hyperplasia,
Includes histological features such as arteriolar stenosis and ischemic injury to the transplanted organ (eg, as determined by biopsy of the graft). In addition, histological signs of renal graft rejection include duplication and thickening of the glomerular basement membrane. Clinical signs of rejection include gradual progression of graft dysfunction. For example, in a subject having a kidney graft, an elevated blood creatinine level may be indicative of rejection. Renal graft dysfunction with normal serum levels of creatinine less than 2.0 mg / dl, most typically about 0.5 to 1.5 mg / dl, is evident by an increase in creatinine levels of about 2-fold above baseline. Become. The presence of anti-donor antibodies and elevated levels of anti-donor antibodies can also indicate graft dysfunction. One skilled in the art will recognize other histological and clinical indicators of rejection.

The present invention also provides a method of screening for an agent that acts synergistically with an immunotoxin in inducing immune tolerance, the method comprising: transplanting a donor graft into a recipient; Administering, thereby reducing the T cell population of the recipient; administering the agent to be screened to the recipient; obtaining a sample containing dendritic cells; then expressing a marker specific for mature dendritic cells Determining the percentage of dendritic cells in the sample that can be induced to express or express, wherein a lower percentage indicates synergy. A "dendritic cell-containing sample" herein can include, for example, a lymph node biopsy, a blood sample, or a tonsillar biopsy. Further, “a marker specific for mature dendritic cells” includes, for example, membrane CD83,
It may include membrane DR, membrane CD86, cytoplasmic P55, or nuclear Rel-B. These can be detected routinely using methods known in the art (O'Doherty et al., 1997).

[0034] Also provided is a method of screening for an agent that inhibits dendritic cell maturation, comprising obtaining a population of immature dendritic cells from a sample containing dendritic cells of interest; the presence of the agent to be screened. Culturing said population of cells underneath; and then determining the percentage of dendritic cells expressing or capable of being induced to express a marker specific for mature dendritic cells, wherein the low percentage is dendritic It shows inhibition of cell maturation. Preferably, the population of cells is cultured in a monocyte-conditioned medium supplemented with TNFα.

The invention also provides a method of treating a subject having an autoimmune disease, the method comprising administering an immunotoxin to the subject, thereby reducing the subject's T cell population;
An agent which inhibits maturation of dendritic cells is administered to a subject. Autoimmune diseases that can be treated by the methods of the present invention include, for example, systemic lupus erythematosus, myasthenia gravis, stiff man syndrome, autoimmune thyroid disease, Sidnam chorea, rheumatoid arthritis. One skilled in the art will recognize various methods for assessing treatment efficacy, including, for example, reducing clinical signs (such as reducing pain, improving muscle function, improving thyroid function) and T cells and / or mature dendritic cells. Histological or biochemical evidence for a decrease in

In the method of the present invention for treating an autoimmune disease, an agent that inhibits maturation of dendritic cells is subjected at least once, or twice, three, four, five, six, seven times to an agent. , 8
Times, 9, 10, 11, 12, 13, or 14 times, preferably for a period of about 2 weeks, and preferably the immunotoxin is administered at least 1, 2 or 3 times, preferably Administer within a period of less than 5 days. As described above, in the context of inducing immune tolerance, when the production of anti-toxin antibody is intended, the immunotoxin is reduced to about 4%.
It can be administered over a period of days. Alternatively, the method of treatment can be further characterized by administering an immunosuppressive agent to the subject. However, treatment with immunosuppressive drugs is of limited duration and can be at lower doses than before.

The immunotoxins and agents that inhibit maturation of dendritic cells described herein, because the immune functions governing graft receptivity and rejection and autoimmune disease are similar among primate species Is believed to be successful in humans for inducing immune tolerance or treating autoimmune diseases.

The present invention also provides a composition comprising an immunotoxin and an agent that inhibits dendritic cell maturation. More specifically, the present invention provides that the immunotoxin is an anti-T cell immunotoxin directed to the CD3 epitope, or the agent that inhibits dendritic cell maturation is N-type.
Compositions that are inhibitors of nuclear translocation of fκB are provided. Preferably, the agent that inhibits dendritic cell maturation comprises one or more NF-AT dependent T
Activates h2 cytokines. Furthermore, agents that inhibit dendritic cell maturation
Preferably, it inhibits one or more NfκB-dependent Th1 cytokines. In one embodiment of the invention, the agent activates one or more NF-AT dependent Th2 cytokines and inhibits one or more NFκB dependent Th1 cytokines. Thus, for example, an agent activates IL4, INF-
γ can be inhibited.

[0039] The compositions of the present invention can further include a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., does not substantially produce an undesirable biological effect, or is included in a pharmaceutical composition. A material that can be administered to a subject with an immunotoxin and an agent that inhibits dendritic cell maturation without interacting in a deleterious manner with the components to be administered. In essence, the carrier is selected to minimize degradation of the active ingredient and minimize adverse side effects in the subject. Suitable carriers can include, for example, water, pyrogen-free saline, pharmaceutically acceptable fats and oils, or mixtures of any of these. The carrier may contain other suitable pharmaceutical additives such as buffers, preservatives, flavors, viscosity or osmotic pressure regulators, stabilizers or suspending agents.

The present invention is described in further detail in the following Examples, which are intended to be illustrative only, and many modifications and variations of the present invention will be apparent to those skilled in the art.

Example 1 Temporary Arrest of Dendritic Cell Maturation to Induce Primate Tolerance In a non-human primate, after induction on the day of transplantation with anti-CD3 immunotoxin,
After depletion of T cells in lymph nodes and blood, stable treatment against MHC mismatched kidney allografts by using a short treatment with the NFκB inhibitor 15-deoxyspergualin to inhibit the proinflammatory cytokine response Good tolerance is preferably achieved. The mechanism of this synergy is a block to maturation of dendritic cell progenitors during the T cell recovery phase after immunotoxin induction.

A normal 3 kg rhesus monkey was given a kidney graft from an unrelated MHC mismatch donor. Monkeys were then given the immunotoxin (10 days) on days 0 and +2.
0 μg / kg / day), methylprednisolone (MP) was administered from day 0 to day +3 with decreasing dose from 7 mg / kg / day to zero, and further from day 0 to day +4 (N = 5) or from day 0 to day +14 (n = 5) treated with deoxyspergualin. Additional recipients include immunotoxin with MP alone (n = 3) or MP and neoral (1
(00 mg / kg / day) was administered on days 0-4 (n = 3).
At days 5, 15, and 30, groin and axial lymph nodes are biopsied and markers of mature dendritic cells, especially membrane CD83, DR, and CD86 and nuclear Rel.
Tested by immunohistochemistry for the presence of -B.

In 80% of IT treatment recipients receiving MP and deoxyspergualin for 15 days, in 40% of recipients receiving MP and deoxyspergualin for 5 days, and MP alone or MP and neoral In 0% of recipients given a renal allograft survived without showing histological or clinical evidence of acute or chronic rejection. The three longest surviving deoxyspergualin animals have immunological evidence of normal renal function and specific tolerance for more than 830 days.

When compared to lymph nodes from transplants without deoxyspergualin, lymph node tissue at day +5 from deoxyspergualin-treated recipients showed an extreme decrease in mature DC, ie, with CD83 membranes Decreased by an average of 58 ± 9.9-fold, 49 ± 9.0-fold for CD86 and 81 ± 13.5-fold for DR. Rel-B nucleus positive cells decreased by 46 ± 7.1-fold. Partial recovery occurred by day 15, and DR +, CD83 +, and Rel-B by day 30.
+ Cells were within the control range. CD86 + cells were also reduced (7.5 ±
1.8-fold reduction). The results show a dose dependent NFκB by deoxyspergualin.
Inhibition and CDp maturation arrest.

The data show that the NFκB inhibitor deoxyspergualin blocks dendritic cell progenitor cell maturation in vivo in non-human primates. we,
We postulate that the unusual synergy between immunotoxin and deoxyspergualin in promoting tolerance in this difficult model is due in part to memory and depletion of naive T cells by immunotoxin. Conceptually, this creates a temporary condition in which the repopulating naive T cells are subjected to a co-stimulatory need for dendritic cells for activation, while dendritic cells The acquisition of costimulatory function of progenitor cells is stopped by deoxyspergualin. The net result is a brief transition to a "non-hazardous" environment similar to that of a newborn baby in a few weeks, while reducing the range of T cells and the maturity of dendritic cell progenitors, This is advantageous for tolerance.

Example 2 To directly examine the maturity of dendritic cell progenitor cells, the isolation of dendritic cells of isolated rhesus monkey peripheral blood in a medium containing monocyte conditioned medium (MCM) supplemented with TNFα was performed. Progenitor cells were examined. Before adding MCM / TNFα, deoxyspergualin (
(0.6-10 mg / ml) was added to the culture of dendritic cell progenitor cells. As in Example 1, expression of the dendritic cell maturation marker was evaluated by flow cytometry and immunocytochemical staining of the cell centrifugation preparation.

In vivo data indicate the transfer of Rel-B staining from the nucleus to the cytoplasm, as well as C
A shift in staining from the membrane of D83 to the cytoplasm was shown, reflecting the phenotype of dominant immature dendritic cell progenitors in deoxyspergualin treated cultures. The in vitro results are consistent with the in vivo results of Example 1.

Example 3 Allograft was carried out in the same manner as in Example 1. Graft recipients were treated with immunotoxin in the form of whole IgG or F (Ab) _{2 on} days 0 and +1 as described in Example 1. Some recipients received DSG daily for 5 days post-transplant or 2 weeks post-transplant. Plasma levels of the Th2 cytokine IL4 and the Th1 cytokine cytokine interferon gamma (IFN-γ) were assayed at one, two, or four weeks after transplantation. After treatment with whole IgG immunotoxin alone, IFN-γ and IL
-4 levels progressively increased from week 1 to week 4, and IFN-γ levels were higher than IL-4 levels. When treatment with whole IgG immunotoxin is combined with DSG treatment, the levels of cytokines change. By two weeks after transplantation,
Clear IL4 polarization is shown in the group receiving the combination treatment. In particular, the group receiving the combination treatment has a substantially lower I compared to the group treated with the immunotoxin alone.
L4 levels were shown, and IL4 levels were substantially lower when compared to IFN-γ levels. The group receiving DSG for 2 weeks showed a sustained and progressive increase in IL4 polarization at week 4, whereas in the group receiving DSG for only 5 days, the polarization was no longer persistent and cytokines were Pattern was reversed. These data indicate that a 2-week course of DSG in combination with
This indicates that it is necessary to achieve a four-polarization angle. IL4 polarization is independent of the type of anti-CD3 immunotoxin, ie total IgG or F (Ab
This occurred regardless of the _two forms. The intensity of the IL4 response was lower for the F (Ab) ₂ form, but the polarization was substantially complete because there was no measurable interferon-gamma. See FIG.

Example 4 Total IgG immunotoxin (FN18-CRM9), F (Ab) ₂ immunotoxin (FN18-
F (Ab) ₂ -CRM9) or phosphate buffered saline (
The RT-PCT method well known in the art for freshly obtained rhesus monkey peripheral blood lymphocytes treated with (PBS) was used. The experimental results are shown in FIG.
FN18-CRM9 induced cytokine gene expression of IL2, IL4, IL10 and INFγ, whereas FN18-F (Ab) ₂ -CRM9 selectively induced IL4
And only IL10 expression was induced. In the presence of low doses of DSG (2.5 μg / ml), induction of IL2, 4, 10 and INFγ by FN18-CRM9 is reduced. In contrast, in the presence of low doses of DSG combined with FN18-F (Ab) ₂ -CRM9, IL4 and IL10 were reduced,
Consistent with in vivo data in Thus, FN18-F (Ab) ₂ -CRM9 in combination with DSG has a unique effect of blocking INFγ expression (consistent with the inhibitory effect of DSG on NF-κB translocation), while IL4 and Promotes expression of IL10. Thus, DS
G differs from cyclosporine or FK506 in that N4 of IL4 and IL10
It preserves and increases activation by F-AT transcription factors, but selectively inhibits the NF-κB-dependent cytokine activation pathway.

Throughout this application, various publications are referenced. The disclosures of these publications are hereby incorporated by reference in their entirety and are not intended to provide a more complete description of the state of the art to which this application pertains. Although the invention has been described with reference to the specific details of certain embodiments of the invention,
Such details should not be taken as limiting the scope of the invention, which is intended to be covered by the appended claims.

Literature 1. Andoins et al. (1996), Transplantation 62: 1543-1549. 2. Posselt et al. (1990), Science 249: 1293-1295. 3. Rermuzzi et al. (1991), Lancet 337. : 750-752. 4. O'Doherty et al. (1997), J. of Immunol. Meth. 207: 185-194.

[Brief description of the drawings]

FIG. 1 shows induction of kidney graft tolerance by treatment of graft recipients with immunotoxin on days 0 and 1 combined with DSG on days 0-14, showing significant Th2. Cytokine polarization occurs. By week 2 post-transplantation, IL4 polarization (lower levels of IL4 compared to the group treated with immunotoxin alone and lower levels of IL4 compared to IFN-γ levels) in the group receiving the combination treatment Is shown. The group receiving DSG over 2 weeks showed a sustained and progressive increase in IL4 polarization at 4 weeks, whereas in the group receiving DSG for only 5 days, the polarization was no longer maintained and the cytokine pattern Was reversed.
IL4 polarization occurred independent of the type of anti-CD3 immunotoxin, ie, total IgG or F (Ab) ₂ form.

FIGS. 2A, 2B, and 2C show whole IgG immunotoxin (FN18-C
RM9), F (Ab) ₂ immunotoxin (FN18-F (Ab) _2- CRM9), or control buffered saline (PBS), performed on fresh rhesus monkey peripheral blood lymphocytes 4 shows the results of RT-PCR. FIG. 2A shows mRNA for IL-2, IL-4, IL-10, IFN-γ in the absence of DSG.
Is shown. FIG. 2B shows IL-2 in cells treated with DSG (2.5 μg / ml).
1 shows mRNAs for IL-4, IL-10, and IFN-γ. FIG. 2C shows the density ratio of various mRNAs to the actin control.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/06 A61P 43/00 105 43/00 105 G01N 33/15 Z G01N 33/15 33/50 Q 33 / 50 Z A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, B , BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO , NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW ( 72) Inventor David M. Neville No. 9624, Parkwood Drive, Bethesda, MD 20814, United States (72) Inventor Judith M. Thomas 2117 F, Brook Highland Ridge, Birmingham, Alabama 35242 United States Tar (Reference) 2G045 AA29 AA40 BB20 BB24 CA18 CB17 CB26 DA54 FA37 JA20 4C084 AA02 AA17 AA20 BA03 BA44 CA62 DC50 MA02 ZB02 ZB21 ZC75 4C086 AA01 DA11 MA02 MA04 NA14 ZB02 ZB21 ZC75 4C206 AA01 AA02 HA31 MA01 MA05 NA14 ZB02 ZB21

Claims (24)

[Claims] 1. An immunotoxin is administered to the recipient, thereby reducing the T-cell population of the recipient; and (b) an agent that inhibits dendritic cell maturation is administered to the recipient. A method of inducing immune tolerance to a transplant in a recipient. 2. The graft is selected from the group consisting of kidney, liver, heart, pancreas, lung, skin, and isolated cell grafts of pancreatic islets, hepatocytes, stem cell progenitor cells, and differentiated stem cell progenitor cells. The method of claim 1, wherein 3. The method of claim 1, wherein the agent that inhibits dendritic cell maturation is an inhibitor of nuclear translocation of NfκB. 4. The method of claim 3, wherein the inhibitor of nuclear translocation of NfκB is an analog or derivative of deoxyspergualin. 5. The method of claim 1, wherein the agent inhibiting the maturation of dendritic cells comprises one or more NFs.
-The method of claim 1, wherein the method activates an AT-dependent Th2 cytokine. 6. The method according to claim 1, wherein the agent inhibiting the maturation of dendritic cells comprises one or more Nf.
2. The method of claim 1, which inhibits a κB-dependent Th1 cytokine. 7. The method of claim 1, wherein the inhibitor of dendritic cell maturation is a soluble IL-17 receptor Fc fusion protein. 8. The method according to claim 1, wherein the inhibitor of dendritic cell maturation is a glucocorticoid. 9. The method of claim 1, wherein the inhibitor of dendritic cell maturation is a tumor necrosis factor alpha binding blocker. 10. The method of claim 1, wherein the inhibitor of dendritic cell maturation is a blocker of granulocyte macrophage colony stimulating factor binding. 11. The method of claim 1, wherein the inhibitor of dendritic cell maturation is a blocker of IL-12p70 binding. 12. The method of claim 1, wherein the inhibitor of dendritic cell maturation is a blocker of interleukin-1β binding. 13. The method of claim 1, wherein the inhibitor of dendritic cell maturation is an anti-CD154 ligand. 14. The method of claim 1, wherein the agent that inhibits dendritic cell maturation is administered to the recipient at least once. 15. The method of claim 1, wherein the agent that inhibits dendritic cell maturation is administered to the recipient prior to transplantation. 16. The method of claim 1, wherein the immunotoxin is an anti-T cell immunotoxin directed to the CD3 epitope. 17. The method of claim 1, further comprising administering to the graft donor an agent that inhibits dendritic cell maturation prior to removing the graft. 18. A method for screening for an agent that acts synergistically with an immunotoxin in inducing immune tolerance, comprising: (a) transplanting a graft of a donor into a recipient; and (b) administering the immunotoxin to the recipient. (C) administering to the recipient the agent to be screened; (d) obtaining a sample containing dendritic cells; and (e) mature dendritic cells. A method comprising determining the percentage of dendritic cells in a sample that expresses or can be induced to express a cell-specific marker, wherein the lower percentage is indicative of a synergistic effect. 19. A method for screening an agent that inhibits maturation of dendritic cells, comprising: (a) obtaining a population of immature dendritic cells from a sample containing dendritic cells of interest; (b) screening Culturing said population of cells in the presence of the agent to be performed;
Then (c) determining the percentage of dendritic cells that express or can be induced to express a marker specific for mature dendritic cells, wherein a low percentage indicates inhibition of dendritic cell maturation The way that is. 20. A composition comprising an immunotoxin and an agent that inhibits dendritic cell maturation. 21. The composition of claim 18, wherein the immunotoxin is an anti-T cell immunotoxin directed to a CD3 epitope. 22. The composition of claim 19, wherein the agent that inhibits dendritic cell maturation is an inhibitor of nuclear translocation of NfκB. 23. The method of claim 1, wherein the agent that inhibits maturation of dendritic cells comprises one or more N
20. The composition of claim 19, which activates an F-AT dependent Th2 cytokine. 24. The method of claim 1, wherein the agent that inhibits dendritic cell maturation comprises one or more N
20. The composition of claim 19, which inhibits an fκB-dependent Th1 cytokine.