CZ403599A3 - Use of agent breaking CD40:CD154 bond for preparing a medicament inhibiting rejection of tissue graft and preparation in which said agent breaking CD40:CD154 bond is comprised - Google Patents

Abstract

Solutions and uses according to the invention are based on the discovery that rejection can be inhibited by the use of an agent abolishing CD40: CD154 binding, either alone or in combination with other immunomodulatory or immunosuppressive agents. For example, a preferred binding agent is a binding-cancellation agent CD4O: CD154 and CD28 Signaling Disruptor. An example of an agent interfering with the defect CD4O: CD154 ummonoclonal antibody antiCD154, eg, antibody-antigen binding characteristics 5c8 monoclonal antibody. An example of a CD28 jamming agent signaling is fusion proteinCILA-Ig. The solution can be made according to the solution use prolonged survival of transplanted tissue in recipient to reverse acute graft rejection and immunologic weakness consequences of graft graft failure.

Description

The present invention relates generally to suppression (suppression) of an undesired immune response, particularly a T-cell mediated anti-adaptive immune response. In particular, the invention relates to the prevention, treatment, suppression or reversal of rejection of a transplanted tissue or organ by a recipient for which the immune system is responsible.

BACKGROUND OF THE INVENTION

Organ transplantation between two individuals that are not genetically identical always results in immunological rejection of an organ caused by a mechanism dependent upon the healing process not being restricted by the administration of drugs that suppress T cell function.

Several US patents, eg, US 5104858, 5008246 and 5068323, disclose the use of immunosuppressive drugs (rejection) in T cells,

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- 2 - _? to inhibit graft rejection. Other conventional agents are described in Suthanthiran et al., 1994, 331, New Eng. Copper. J. 365-376. Both calcineurine phosphatase inhibitors and glucocorticosteroids are used clinically and both agents prevent T cell-mediated release of activating cytokines, especially IL-2. However, therapy with this type of conventional agent is imperfect. Both types of agents act by disrupting signaling via the T cell antigen receptor (TCR), which. is the only mediator of antigen specificity of T cells, and thus they act on all T cells indiscriminately. In addition, the effect of these drugs is not sustained, so discontinuation of treatment generally results in graft loss. Thus, in order to maintain a viable graft and its functional integration, the transplant recipient must suffer the consequences of long-term non-specific immunosuppression. These consequences include increased risk of infection and malignancies, toxicity as well as significant costs.

Thus, there is a need for improved or more effective treatment by immunosuppression or immunomodulation in transplant recipients. In particular, a treatment that does not lead to general T-cell suppression, i. does not increase the susceptibility of the recipient to malignancies or opportunistic infections. Above all, however, there is a need for a treatment that is less toxic than the currently used therapeutic agent. . At the same time, there is a need for treatment that promotes long-term functional graft integration, i.e., integration that persists in discontinuation of treatment.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an immunomodulatory agent that attenuates the anti-adaptive T cell response without leading to a general immunosuppression of the T cells. Another object of the invention is an immunomodulatory agent that promotes functional integration of a tissue graft in a recipient. Another object of the invention is an immunomodulatory agent that inhibits immunological rejection of transplanted tissue. Another object of the invention. is an immunomodulatory agent that disrupts the transmission of a costimulatory signal to activated T cells. In particular, the present invention provides a CD40: CD145 binding disruptor for use in therapy, particularly for use in treating or delaying immunological rejection of transplanted tissue. Another particular object of the present invention is a therapeutic composition and treatment regimen for attenuating a T cell-mediated anti-adaptive immune response based on the use of a CD40: CD145 binding breaker in combination with another immunosuppressive or immunomodulatory agent. Thus, it is a specific object of the invention to provide a therapeutic composition and treatment regimen based on the use of a CD40: CD145 binding debonding agent (binding breaker) in combination with another agent that blocks costimulation by CD28. In general, it is an object of the invention to provide a therapeutic composition and treatment regimen for inhibiting, alleviating, attenuating, delaying or reversing (reversing) failure or acute rejection of transplanted tissue. In general, it is a further object of the invention to improve the viability of a tissue graft by providing an immunomodulatory composition that allows the recipient to functionally integrate allogeneic or xenogeneic tissue (allograft or xenografts). Furthermore, it is generally an object of the invention to prevent, alleviate, or treat a disease resulting from an anti-adaptive immune response, including T-cell mediated autoimmune diseases (eg, insulin-dependent diabetes mellitus, multiple sclerosis, and the like) and allergies.

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The present invention is based on the discovery that the use of a CD40: CD154 binding debonding agent, alone or in combination with other immunomodulatory agents, attenuates, inhibits, prevents, delays or abolishes anti-adaptive rejection of transplanted tissue by the host immune system without the need for overall suppression. the recipient's immune system.

Accordingly, the present invention provides the use of a CD40: CD154 binding debonding agent for the preparation of a therapeutic composition and proprietary compositions for immunomodulatory therapy in recipients of tissue grafts (transplants). These compositions can be used in a variety of therapeutic methods (hereinafter, methods of the invention). In a first method of the present invention, graft rejection is inhibited by administering to the recipient a CD40: CD154 binding agent (CD40L). A debonding agent, a breaker, is any agent that destroys the costimulatory molecule (herein a CD40 ligand, also referred to as an antigen to its counterpart, CD40). Preferably it is

5c8, CD40L, CD154, and sometimes also gp39) or a recognized receptor (herein, an anti-CD40L compound interrupter), meaning a compound that binds to CD40L (CD154) and thereby blocks or abolishes the ability of CD40L to bind to CD40. CD40L is a monoclonal antibody, in particular a monoclonal antibody that has antigen binding characteristics identical to antibody 5c8 described in US 5474771, the disclosure of which is hereby incorporated by reference.

The second method of the present invention prolongs graft survival in the recipient by administering to the recipient a CD40: CD154 binding breaker, preferably an anti-CD40L monoclonal antibody. .

A third method of the invention attenuates immunological complications or transplant tissue failure by administering to the recipient a CD40: CD154 binding breaker, preferably an anti-CD4QL monoclonal antibody. That is, the method results in inhibition, suppression, amelioration or detectable reduction of immunological problems. Specifically, the method of the invention completely prevents or attenuates complications such as interstitial fibrosis, chronic atherosclerosis, graft vasculitis, and the like.

The methods of the invention mentioned above are effective for treating acute and / or chronic tissue graft rejection and can be used prophylactically, for postoperative treatment or for reversing or suppressing graft rejection at any time in the recipient's life. One of the methods of the invention, exemplified, is to administer a CD40 debonding agent: CD154 2, 4, 6, 8, 12, 16, and 28 days after surgery. In general, the methods of the invention comprise administering the interfering agent at the desired intervals (daily, twice weekly, weekly, once every 2 weeks, etc.) for at least two to three weeks. The administration schedule is adapted to result in a detectable reduction in the symptoms of an anti-adaptive immune response, particularly the symptoms of graft rejection. Therapeutic regimens may be repeated for other episodes of graft rejection. In embodiments of the present invention, wherein. the interfering agent is a monoclonal anti-CD40L antibody, administered at doses of 5 mg / kg to 20 mg / kg body weight.

For use in treating a CD40: CD154 binding agent is formulated in a therapeutic composition comprising a therapeutically effective amount of a binding agent dispersed in a pharmaceutically acceptable carrier. In some embodiments of the present invention, the therapeutic composition comprises a therapeutically effective amount of other immunosuppressive or immunomodulatory compounds including, but not limited to: a costimulatory interfering agent

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T cell signaling via CD28 (eg CTLA4-Ig), a calcineurin signaling disrupter (eg cyclosporine, macrolides such as tacrolimus, formerly known as FK506), corticosteroids or antiproliferative agents (eg azathioprine). Other therapeutically active compounds suitable for use with the CD40 binding agent: CD154 of the invention include sirolimus (formerly known as rapamycin), mycophenolate mofetil (MMF), moziribine, deoxyspergualin, sodium brequinar, leflunomide, azaspiran and the like.

The compositions of the present invention are suitable for use in all cases of graft transplantation. Thus, the invention is useful for use where the graft recipient is a mammal, preferably a primate, most preferably a human. The graft donor is a syngeneic member of the same phylogenetic species as the recipient (i.e., an allogeneic donor that provides a tissue allograft) or a member of a different phylogenetic species (i.e., the donor is xenogenic, provides a tissue xenograft). If a xenogeneic donor is used as the source of the transplanted tissue, it is preferably relatively MHC-compatible with the recipient, eg a baboon or chimpanzee would be the preferred donor to humans. The invention preferably promotes graft removal of any tissue or organ, regardless of organ _t or (non-limiting) whether the donor graft is an entire organ, part of a tissue or isolated. cells. Examples of suitable tissues are tissues. kidney, liver, heart, pancreas (eg islets), skin, blood vessels, nerves, bones, cartilage and similar mammalian tissues.

The principle of the present invention has been verified by testing on a relevant precliriic model. One example of a CD40 binding agent: CD154 (namely anti-CD40L monoclonal antibody 5c8) was tested alone or in combination with other immunomodulators (eg, CD28 CTLA4-Tg binding agent, mycophenolate mofetil, corticosteroids, tacrolimus)

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on rhesus (Macaca mulata) peripheral blood leukocytes in vitro and then directly on the macaques with primarily vascularized renal allograft.

Other objects of the invention, their features and advantages, as well as. invention. alone, will be described in detail in the following and in the form of preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

Over the past 20 years, data has accumulated showing that T cell activation requires both TCR-mediated signals and simultaneously transmitted costimulatory signals. For example, antibody production by B cells in response to protein antigens requires specific, costimulatory interaction by T cells. Such interaction of B cells with T cells is mediated, in addition to TCR participation, by several receptor-ligand binding events. These additional binding events include the binding of CD40 on B cells to CD154 (CD40L) on

T cells. Human CD40 is a 50 kD cell surface protein expressed on mature B cells, as well as on macrophages and activated endothelial cells. CD40 belongs to a class of receptors involved in programmed cell death (apoptosis), including Fas / CD95 and the tumor necrosis factor (TNF) alpha receptor. Human CD154 (CD40L) is a 32 kD type II membrane glycoprotein that exhibits homology to TNF alpha, which is transiently expressed primarily on activated T cells. CD40: CD154 binding was shown to be necessary for all types. T cell dependent antibody responses. In particular, CD40: CD15 binding provides an anti-apoptotic and / or lymphokine stimulation signal.

Another important costimulatory signal is the binding of CD28 on T cells to its counterpart, CD80 (B7-1) or CD86 (B7-2) receptors on antigen-presenting cells (APS) and perhaps

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9 99 also on parenchymatic cells. CD80 and / or CD86 expression is increased by signals that initiate CD40: CD154 binding. Further studies have shown that CTLA4 (CD152) T cell molecules reduce costimulation by TCR-mediated activation, at least in part by competing with CD28 for CD80 / CD86 and by transmitting a unique negative TCR signal to the signal transduction complex.

The importance of CD40: CD154 binding in promoting T cell dependent biological responses is underlined by the development of X-linked hyper-IgM syndrome (X-HIGM) in humans lacking functional CD154. These individuals have. normal or high IgM levels, but IhG, IgA or IgE antibody production fails. Affected individuals suffer from repeated, sometimes very severe, bacterial infections (most commonly Streptococcus pneumonia and Haemophilus influenzae) and some unusual parasitic infections, as well as an increased incidence of lymphomas and abdominal cancers. These clinical manifestations of the disease can be managed by therapy where intravenous immunoglobulin is replaced.

Effect of disease X-HIGM. is simulated in animals nullizygous for the gene encoding CD154 (knockout) Studies with nullizygous animals have confirmed that while B cells can produce IgM even in the absence of CD40: CD154 binding, they are not capable of isotype switching under such conditions ( isotype switching) or survive normally after affinity maturation. In the absence of a functional CD40: CD154 interaction, the germinal lymph node centers do not develop properly and memory B cell development is impaired. These defects then contribute to a strong reduction or complete disappearance of the secondary (mature) antibody response.

_f Individuals with X-hIgM and CD154 nullizygous individuals also suffer damage to cellular immunity. Such defects are manifested by an increased incidence of Pneumocystis carinii, Histoplasma capsulatum, Cryptococcus neoformans-

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chronic infection with Giardia lambli. Nullizygous mice are unable to resist Leishmania infection. Many of these cell-mediated defects are reversible, i. can be removed by administration of IL-12 or IFN-gamma. These data support the view that CD40: CD154 binding promotes the development of a Type I T helper cell immune response. Further support is the observation that macrophage activation is defective in CD154 deficiency and that administration of anti-CD40L antibodies to mice reduced their ability to suppress Pneumocystis infection. Blocking CD40: CD154 binding reduces the ability of macrophages to form nitric oxide, which mediates many of the pro-inflammatory activities of macrophages. It should be noted that mammals, including humans, lacking functional CD154 do not show an increased incidence of viral infections or sepsis.

Numerous preclinical studies have found that agents capable of disrupting CD40: CD154 binding are promising immunomodulatory agents. In murine antibodies to CD154, both primary and secondary immune responses to exogenous antigens block both in vitro and in vivo. Antibodies to CD154 cause a reduction in the germinal centers of mice and monkeys, in accordance with CD154 immunodeficiency data. Administration of three doses of anti-CD154 'antibody to lupus-prone mice at 3 months of age significantly reduced antibody titer against double-stranded DNA and nucleosomes, slowed the development of severe nephritis and reduced mortality. Moreover, administration of anti-CD154 antibody to mice aged five to seven months with severe nephritis has been shown to stabilize or even reverse the course of kidney disease. AntiCD154 antibody, co-administered with small resting allogeneic lymphocytes, allowed unrestricted survival of pancreatic islet grafts in mice. In other animal models, interference with CD40: CD154 binding has been demonstrated to reduce symptoms of autoimmune disease (e.g., multiple sclerosis, rheumatoid arthritis).

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- 10 arthritis, inflammatory bowel disease), graft rejection (allograft heart, graft-versus-recipient disease) and mercuric chloride-induced glemerulonephritis, which is mediated by both humoral and cellular mechanisms.

Further rodent studies have shown that T cell activation can be blocked, thereby prolonging allograft survival, by affecting CD80 / CD86 binding to the respective T cell receptor, CD28 and CTLA4. In these studies, the CD80 / CD86 specific CTLA4-Ig fusion protein was used as a CD28 signaling interrupter. Other experiments have shown that an increase in CD80 / CD86 - can be prevented by using a CD40: CD154 binding debonding agent (eg, MR1 monoclonal antibody). The efficacy of both classes of immunomodulatory agents is dependent on TCR participation. Such agents offer the capacity to modulate the specificity of T cell dependent biological processes rather than total T cell immunosuppression. Studies using such agents in vivo in rodent graft rejection models have shown dramatic results, including the acceptance of a completely incompatible skin graft, a result that cannot be achieved with currently available immunosuppressants.

However, it is worth noting that all previously published studies of long-term graft survival in rodents have failed or have been associated with unacceptable toxicity when tested in other animals, especially primates.

On the other hand, the described baseline validation study found that the CD40: CD154 binding debonding agent alone or in combination with other immunomodulatory or immunosuppressive agents (such as the CD28 signaling interrupter) promotes long-term integration of heterologous donor tissue without rejection by the primary recipient. It is encouraging that the therapy described in the invention is fairly simple, including administration of the therapeutic agent

·· · ··· standard peripheral intravenous catheter, and has been excellently cleared by the recipient, regimens that

This is in marked contrast to others used for long-term graft acceptance in primates and which require ionizing radiation irradiation, donor-derived bone marrow administration and marked overall immunosuppression around the operation. The animals treated in the study described in the invention did not show any signs of T cell activation or cytokine release usually seen after treatment with anti-CD3 antibodies, and prolonged survival did not entail increased costs in terms of treating opportunistic infections. In addition, no changes in peripheral blood hematological parameters were found. Long-term survival was achieved without apparent removal or total reduction of any lymphocyte subpopulation and without. loss of T cell responsiveness in vitro. Therefore, the effects observed are unlikely to result from destruction of T cells after opsonization with antibody or. fusion protein. The results are surprising. Such success in outbred rhesus rhesus monkeys leads to the conclusion that the integration of allograft (and even xenograft) is an achievable target in humans if the method of the invention or its equivalent is used.

Furthermore, the mechanism of action and the relative contribution of each agent in the combination therapy described remains unclear. The success of CD40: CD154 blocking alone suggests that any basal costimulatory signaling is less important for maintaining the healing response than regulation by increasing CD80 / CD86. Indeed, administration of anti-CD154 antibody alone led to surprising survival without healing, whereas the effect of the CD28 breaker (CTLA4-Ig) was rather transient. Since CD154 is expressed on non-myeloid cells, including vascular endothelium and smooth muscle, and CD80 can be induced on fibroblasts and hepatocytes, events

Non-T cells related to non-T cells may be critical for developing reactivity against donor tissue. By preventing the immune system from accessing significant parenchymal and costimulatory signals at the time of transplantation, it is possible to prevent the recognition of the steppe and its destruction. Differences in donor parenchyma-induced activation and lymphoid cell-induced activation could explain the observed maintenance of in vitro reactivity to donor lymphocytes despite normal graft function and thus generally poor correlation between MLR reactivity and clinical graft behavior. However, the action of selected costimulatory blocking agents CTLA4-Ig and humanized 5c8 (anti-human CD154) was shown to be synergistic both in vitro and in vivo. Perhaps CTLA4-Ig provides a safeguard against the expression of CD80 / CD86 that escapes the effect of disrupting CD40: CD154 binding by humanized 5c8. In this case, considerable time is needed to show acute rejection with only a few remaining cells that have escaped the initial block.

Since this strategy was effective in averting the already established, biopsy-proven healing, it is clear that the healing process is maintained by continuous costimulation rather than being a process that, once initiated, continues until effector cells are eliminated or discarded TCR signaling. Teleologically speaking, the body benefits most from inflammation, which is easily controlled. So if the direction of the attack is not clear, retreat can be an unspoken order. This supports the view that the use of the natural tendency of the immune system to downregulate could be preferable to overall immunosuppression.

The following discussion demonstrates a number of circumstances in which the invention may be used, and the examples then describe the basic principle demonstrating the study and specific embodiments of the invention.

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Recipients

The invention can be used in the treatment or prophylaxis of any recipient of a tissue graft or any mammal in need of a transplant. Preferably, the recipient is referred to as a host or only a primate, more preferably a higher primate, and most preferably In another embodiment, the recipient is another in need of a tissue graft, particularly a commercially significant animal species, or companion pet or other valuable animal such as . Thus, suitable recipients include, but are not limited to, sheep, horses, cows, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice.

Tissue graft donors

The invention can be used in any kind of tissue transplantation or transfer, particularly where the donor tissue (graft) is at risk of being healed by the recipient's immune system. In particular, the invention can be used where the donor tissue is not histocompatible with the recipient. Thus, in addition to autologous or syngeneic donor tissue, the invention may be used for allogeneic or even xenogeneic donor tissue. The donor tissue may be obtained in a conventional manner from voluntary donors or other living donors, or it may be obtained from a deceased donor (i.e. from a cadaver). Preferably, the donor is histocompatible with the recipient as much as possible. If the recipient is a human, the preferred donor tissue is autologous and allogeneic. However, donor tissue can be obtained from a heterologous species (in this case referred to as a hetero-graft) such as from non-primate primates

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000 00 00 human (chimpanzee or baboon) or another ^? a relatively compatible animal (pig).

In some embodiments, the donor tissue (graft, transplant) is an entire organ or body part. In other embodiments, the graft comprises a portion or biopsy of a donor organ or tissue. In yet other embodiments of the invention, the donor tissue is cells, particularly isolated cells or suspended cells, including cells excised or excised from the donor, cells maintained in primary culture, or cells immortalized in cell lines. Optionally, the donor tissue may comprise cells carrying exogenous genetic material, such as transfected or transformed host cells, which have been (or are derived from, those cells that were originally) engineered to contain the genetic material necessary for production. a polypeptide having therapeutic significance for the recipient. In other embodiments of the invention, the donor tissue may be obtained from transgenic mammals that have been engineered by genetic engineering to contain, in some or all of the body tissues, the genetic material necessary to produce a polypeptide of therapeutic interest to the recipient. For examples of such therapeutically important polypeptides include hormones such as insulin or growth hormone, cytokines, growth and differentiation _factors,> enzymes, structural proteins and others.

Thus, in light of the foregoing, it is clear that the invention can be used for transplantation of solid organ grafts such as kidney, liver, pancreas, lung, heart and the like. other types of tissues, particularly kidney, liver, pancreas (especially islets), respiratory tissue, tissue of the heart, skin, blood vessels, nerves, bones, bone marrow, cartilage, tendons,

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- 15 ligaments, muscles, adipose tissue, breast tissue, gastrointestinal lining, epithelium, endothelium, connective tissue, etc. In addition, the invention may be applied to parts of the body that contain multiple tissue types, such as a surrogate or other type of surgical alteration, or reconstruction of the eye, ear, nose, finger, joint, blood vessel, nerve, muscle, limb or other part of the body.

In other embodiments, the invention can be used for cell preparations or suspensions introduced systemically or locally into the recipient. For example, isolated, suspended, or dispersed cells may be. administered by intravascular infusion or implanted at a desired site such as a bone marrow cavity, liver, renal capsule or articular capsule, or may be administered intramuscularly or locally to the injured site. Examples of such cells include peripheral blood cells, bone marrow cells or any hematopoietic cells, mesenchymal stem cells, muscle satellite cells, hepatocytes, hormone producing or neuroendocrine cells, fibroblasts, nerve crested cells, endothelial cells and the like. In some embodiments, the cells are mitotically competent and create new tissue of donor origin. In other embodiments, the cells are not mitotically competent, but produce or express a polypeptide or other product of therapeutic interest to the recipient.

Examples of CD40 debonding agents: CD154

Therapeutic compounds suitable for the methods of the invention include any compound that blocks the interaction of CD40 on the cell surface (e.g., B cells) with CD40L (CD154) expressed on the surface of activated T cells. Compounds that disrupt CD40: CD154, such as an anti-CD40L compound that are specifically included in the invention, are e.g.

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polyclonal antibodies and monoclonal antibodies (mAbs) as well as antibody derivatives such as chimeric molecules, humanized molecules, molecules with reduced effector function, bispecific molecules and antibody conjugates. In a preferred embodiment of the invention, the antibody is 5c8 as described in U.S. Pat. In a currently highly preferred embodiment of the invention, the antibody is a humanized antibody 5c8. Other known anti-CD154 antibodies include ImxM90, ImxM91 and ImxM92 (from Immunex), the monoclonal antibody CD40L commercially available from Ancell (clone 24-31, catalog # 353-020, Bayport, MN), and the monoclonal anti- CD40L commercially available from Genzyme (Cambridge, MA, Catalog No. 80-3703-01). Also available is a commercially available anti-CD40L monoclonal antibody from PharMingen (San Diego, Catalog No. 33580D). Many other anti-CD40L antibodies have been prepared and characterized (see, eg, WO 96/23071, BristolMyers Squibb, the disclosure of which is hereby incorporated by reference).

The invention also relates to other anti-CD40L molecules of another type, such as Fab, F (ab ') ₂ fragments, V _H , Fv regions, single chain antibodies (see eg WO 96/23071), polypeptides, polypeptide fusion constructs, fusions containing CD40 (such as CD40Ig, see Hollenbaugh et al., J. Immunol. Meth. 188: 1-7, 1995) and small molecule compounds such as small semipeptide compounds or non-peptide compounds capable of blocking or disrupting CD40 binding : CD154. Methods for designing, screening, and optimizing small molecules have been described in PCT / US96 / 10664, filed Jun. 21, 1996, which is hereby incorporated by reference.

Various forms of antibodies can be prepared by standard recombinant DNA techniques (Winter and Milstein, Nature 349:

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293-99 (1991). E.g. chimeric antibodies can be constructed in which the antigen-binding domain of an animal antibody is linked to a human constant domain (the antibody originated originally from a non-human mammal, where recombinant DNA technology has been used to replace all or at least a portion of the hinge region and heavy and / or the light chain by corresponding regions from a human immunoglobulin heavy or light chain (see, eg, Cabilly et al., U.S. Patent 4,816,567, Morrison et al., Proc. Natl. Acad. Sci. USA 81; 6851-55, 1984). Chimeric antibodies reduce the immune response elicited by an animal antibody when used in human clinical treatment.

In addition, it is possible to synthesize humanized recombinant antibodies. Humanized antibodies are those originally derived from a non-human mammal in which recombinant DNA technology has been used to replace some or all of the amino acids that are not required for antigen binding with other amino acids from the corresponding light and heavy chain regions of a human immunoglobulin. Or they are chimeras containing, for the most part, human immunoglobulin. sequences into which regions responsible for specific antigen binding have been inserted (see, eg, PCT Patent Application WO94 / 04679). Animals are immunized with the desired antigen, the corresponding antibodies are isolated, and the portion of the variable region responsible for specific antigen binding is removed. The antigen binding regions from the animal are cloned into appropriate sites in the human antibody genes from which the antigen binding regions have been removed. Humanized antibodies minimize the use of heterologous (cross-species) sequences in antibodies for use in human therapy, and thus reduce the likelihood of causing an undesired immune response. Similarly, primatized antibodies can be generated. ,

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Another embodiment of the invention involves the use of human antibodies that can be prepared in animals, eg, in transgenic animals that carry one or more human immunoglobulin transgenes. Such animals can be used as a source of splenocytes for the preparation of hybridomas, as described in U.S. Patent No. 5,569,825.

Antibody fragments can also be used in the methods and compositions of the invention. and univalent antibodies. Univalent antibodies comprise a heavy / light chain dimer bound to the Fc region (stem) of the second heavy chain. The Fab region refers to a chain region that. is roughly equivalent or analogous to a sequence that comprises a heavy chain region from the Y-branched region and the entire light chain, and which (in aggregates) exhibits antibody activity. Protren Fab contains heavy and light chain aggregates (commonly known as Fab ') as well as tetramers that correspond to branched Y-shaped antibody segments (known as F (ab') 2) z whether they are aggregated covalently or non-covalently if the aggregates are capable selectively react with a particular antigen or antigenic family.

In addition, standard recombinant DNA techniques can be used to alter the binding affinity of recombinant antibodies to their antigens by altering amino acid residues near the antigen binding sites. The antigenic binding affinity for humanized antibodies can be increased by mutagenesis based on molecular modeling (Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-33, 1989, PCT patent application WO 94/04679. reduce the binding affinity of antibodies to CD40L, depending on the type of target tissue or the particular treatment scheme envisaged, which can be done using a phage display tehnique technique, see, eg, Winter et al., Annu.

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Roar. Immunol. 12: 433-445, 1994 and Schier et al., J. Mol. Biol. 255: 28-43, 1996, incorporated herein by reference). E.g. it may be advantageous to administer to the patient a constant level of antibodies with reduced affinity for CD40L for semiprophylaxis. Alternatively, antibodies with increased affinity for CD40L may be advantageous for short-term treatments.

Routes of administration

The compounds of the invention may be administered by any medically acceptable route. Depending on the specific circumstances, local or systemic administration may be desirable. Preferably, the compound of the invention is administered parenterally, e.g., intravenous, intraarterial, subcutaneous, intramuscular. intraorbital, intraventricular, intraperitoneal, subcapsular,, intracranial, intraspinal or intranasal injection, or by infusion or inhalation. The compound may also be administered by biocompatible or sustained release implantation, either before or after implantation of the donor tissue, by infusion pump or bioerodible imolant implant. Alternatively, certain compounds of the invention or compositions comprising them are suitable for oral administration or enteral administration. Other formulations of the invention are suitable for topical administration.

In general, the compounds of the invention are administered to a transplant recipient. However, these compounds can also be administered to the donor and / or into the donor tissue. E.g. the compound of the invention may be part of a perfusion or preservative solution in which the transplanted tissue is stored or in which the tissue is transported prior to transfer to the donor.

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Dosage and frequency of administration

The size and frequency of dosages of any particular compound to be administered to a patient with a given immune complex disease is within the competence and clinical judgment of a particular attending physician trained in the field of transplantation, such as a transplant surgeon. The general dosage and administration regimen is determined based on preclinical and clinical studies, which include extensive but routine studies to determine optimal parameters for administration of a given compound. Even after these general guidelines are provided. administration, the attending physician will often vary the dosages for different recipients based on a number of considerations such as age, health, weight, sex, and optionally treatment with other agents in a particular individual.

Determining the optimal dosage and administration regimen for each of the anti-CD40L compounds that are used to inhibit graft rejection is routine to those of skill in the pharmaceutical and medical arts.

Generally, dosages are determined by the attending physician or other similarly experienced physician, and the dosing regimen may consist of periods of high frequency, such as daily or weekly intervals, alternating with periods of less frequent administration. such as once a month or even less often.

To give an example of dosing an anti-CD40 compound, we present the dosing strategy of the anti-CDL40 monoclonal antibody. Dosages can accordingly be readily determined for other types of anti-CD40L compounds. In general, a single dose of 0.05 to 50 mg / Kg of body weight of the patient is contemplated, with doses of 1 to 20 mg / kg being the most common. For acute treatment, for example, before or directly at the time of transplantation, or when symptoms suggest that healing has begun, fib

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The effective dose of the antibody is 1 mg / kg to 20 mg / kg body weight administered daily after. for 1 to 5 days, preferably by bolus intravenous form. The same dosages and dosing schedule can be used in a load regimen and a maintenance regimen wherein the maintenance phase comprises intravenous or intramuscular administration of the antibody in an amount of 0.1 mg / kg to 20 mg / kg body weight, for treatment from weekly to 3 month intervals. . Chronic administration may also be performed in a maintenance regimen wherein the antibody is administered by the intravenous or intramuscular route at a dose of 0.1 mg / kg to 20 mg / kg body weight, with intervals between 1 week to 3 months. In addition, chronic treatment may be performed in an intermittent boius intravenous regimen, whereby an antibody dose of 1.0 mg / kg to 100 mg / kg body weight is administered with intervals between successive doses of 1 to 6 months. In all regimens except for the intermittent bolus, the compound may also be administered by the oral, pulmonary, nasal or subcutaneous routes.

According to the 'alternative. Embodiments of the invention the efficacy of antibodies in inhibiting graft rejection can be enhanced by serial administration or by administration in combination with conventional therapeutic agents or anti-rejection drugs such as corticosteroids or immunosuppressants. Alternatively, the antibodies may be conjugated to conventional reagents. This preferably allows the administration of conventional agents in an amount that is less than the usual dose, e.g., less than 50% of the conventional dose, if the agent were to be administered in monotherapy. Thus, many side effects of conventional agents can be avoided by this method.

Combination therapies of the invention for the treatment of graft rejection include the use of anti-CD40L antibodies together with B cell targeting agents such as antibodies.

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22 anti-CD19, anti-CDE28 or anti-CD20. (unconjugated or radiolabeled), IL-14 antagonists, LJP394 (receptor blocker, LaJolla Pharmaceuticals), IR-1116 (small molecule, Takeda) and anti-Ig idiotypic monoclonal antibodies. Alternatively, the combinations may include T cell / B cell targeting agents such as CTLA4Ig, IL-2 antagonists, IL-4 antagonists, IL-β antagonists, receptor antagonists, α-CD80 / CD86 monoclonal antibodies, TNF, LFA1 antagonists / ICAM, VLA4 / VCAM antagonists. brequinar-IL-2 toxin conjugates (eg DAB), prednisone, anti-CD3 monoclonal antibody (OKT3), mycophenolate mofetil (MMF), cyclophosphamide and other immunosuppressants such as calcineurin signaling blockers, including but not limited to tacrolimus (FK506). The combination may also include agents directed to T cells, such as CD4 antagonists, CD2 antagonists, and IL-12.

To maintain graft integration, or in the period following suppression of the acute graft rejection episode, a maintenance dose of anti-CD40L antibodies is administered, either alone or in combination with conventional anti-rejection drugs, if necessary. Later it is possible to reduce the dose or dose frequency, or both. If the symptoms of healing have disappeared, treatment may cease, but close monitoring of healing symptoms is required. In other cases, as determined by the skilled practitioner, an intermittent administration regimen may be determined, eg, at intervals of four weeks or longer. However, the recipient may require intermittent treatment in the long term if symptoms of the disease reoccur.

Preparations

In general, the compounds of the invention are suspended, dissolved or dispersed in a pharmaceutically acceptable,

G · · ·

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9 9 9

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23 carriers or excipients. The resulting therapeutic composition does not adversely affect the recipient's homeostasis, particularly the electrolyte balance. For example, a suitable carrier comprises normal saline (0.15M NaCl, pH 7.0 to 7.4). Other acceptable carriers are well known to those skilled in the art and are described in the literature, e.g., Remington's Pharmaceutical Sciences, Gennaro (ed), Mack Publishing Co., 1990. Acceptable carriers also include carriers containing biocompatible, inert, or bioabsorbable salts, buffering agents, oligo. - or polysaccharides, polymers, viscosity-adjusting agents, preservatives and the like.

The anti-CD40L compound of the invention is administered in a pharmaceutically effective or therapeutically effective amount, an amount that is capable of eliciting a detectable, preferably medically beneficial, effect in a recipient that is expected or already suffering from graft rejection. Medically beneficial effect means preventing, delaying or alleviating the deterioration of the health condition or a noticeable improvement in the health status of the recipient. E.g. for the indication of renal allograft or xenograft status, the health and renal function are monitored by one or more routine laboratory tests that measure the concentration of relevant substances in blood or urine, or other urine characteristics, or the clearance rate of various substances from blood and urine. Parameters obtained. In these tests, either alone or in combination, the attending physician will use it to assess renal function or damage. Examples of endpoints include blood urea, creatinine or protein concentration, protein or various blood cell concentrations such as urinary erythrocytes or leukocytes, urine density, urine amount, insulin clearance rate, creatinine, urea or aminohippuric acid, or the presence of hypertension or edema.

9 9 9 9 999 999 999

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- 24 -.

As a specific example of the use of the invention in clinical practice, administration of an anti-CD40L monoclonal antibody to renal donor recipients may be mentioned. tissue at the same time as surgery or after the appearance of graft rejection symptoms. Acute renal allograft rejection is manifested by a number of symptoms, including an increase in serum creatinine and blood urea nitrogen, a decrease in urine output, the development of proteinuria and / or haemouria, and others. The amount and time course of immunotherapy administered should be sufficient to cause clinically beneficial changes in one or more of these symptoms. An example of time course of immunotherapy and dosing is given in the evidence study included in the examples. Therapy necessarily involves administering a CD40: CD154 binding agent (e.g., hu5c8) intravenously at a bolus dose of up to 50 mg / kg, followed by an appropriate follow-up regimen _: (e.g., daily intravenous or subcutaneous injection) for several weeks from initiation of therapy or it is not apparent that favorable changes in the symptoms of rejection or graft failure have been achieved.

Another example is the administration of an anti-CD40L compound, in a similar manner as described above, to a recipient with symptoms of other graft rejection. E.g. acute rejection of transplanted liver. leads to jaundice (hyperbilirubinaemia), hepatitis (elevated levels of aminotransferases), coagulantopathy and encephalopathy.

Preclinical model systems for the evaluation of treatment regimens using a CD40: CD154 binding debonding agent

An example of a preferred model system for testing the efficacy of a CD40 binding agent: CD154 (eg, an anti-CD40L compound such as monoclonal antibody 5c8) is the primate renal allograft model described in the provisional US Patent Application S.N. 60/049 389 (06/11/97) and Kirk.

 Et al., 1997. Proc. Nati. Acad. Sci. USA 94: 8789-8794, the disclosure of which is incorporated herein by reference). The described rhesus macaque model (Macaca mulata) has repeatedly been shown to be a very rigorous model for immunity testing, is extremely sensitive to slight changes in allograft function or adverse changes in wound healing in the recipient or immune system function. In addition, it shows an apparent biological similarity to human kidney transplantation. In particular, genes that encode major histocompatibility complex (MHC) proteins are very conserved when comparing cynomolgus monkeys and humans and rejection of vascularized organs is parallel to what is observed clinically.

This model system is appreciated as a preferred system for evaluating grafts containing renal tissue. However, other preclinical model systems known in the art, preferably primates, are useful for assessing the efficacy of other tissue grafts such as liver, heart, lung, pancreas, islets, pancreas, skin, peripheral or central infiltrate, or other types of tissues or organs.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Material and methods

Reagents

Human CTLA4-Ig and control IgG1 fusion protein were prepared and delivered in solution by Genetics Institute, Cambridge, MA. The anti-CD40 ligand, humanized 5c8, was prepared and delivered in solution by Biogen

 «· · · · 9 · ř» 9 · · ·

6.

Corporation, Cambridge, MA. Hamster monoclonal anti-mouse CD28 antibodies PV1 (IgG1, clone G62) were purified from hybridoma culture supernatant and used as isotype control monoclonal antibodies.

MHC typing and donor / recipient selection

The donor-recipient and animal combination selected for third-party foreign cells was selected on the basis of non-identity genetic diversity in both I. and II. MHC class. Class I MHC mismatch was assessed by one-dimensional isoelectric focusing and II mismatch. class MHC was determined based on the results of unidirectional mixed lymphocyte (MLR) reactions. In addition, the diversity of DRB loci of animals was confirmed by denaturing gradient gel electrophoresis and then by direct sequencing of the second DRB exon. The strong responsiveness of the recipient T cells to the donor was confirmed in vitro for all selected donor-recipient pairs · All experiments described herein were performed in accordance with the rules given in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animals Resources, National Research Council, DHHS, Publication No. NIH 86-23, 1985.

In vitro cell analyzes

One-way MLR assays were performed on all pre-transplant animals and all animals. surviving transplanted animals without symptoms of healing after 100 days. Each animal was tested against all potential donors to select the most matched pair. The corresponding cells (3 x 10 ⁵ ) were incubated with irradiated cells (1 x 10 ⁵ ) at 37 ° C for 5 days. Cells were pulsed with ³ H-thymidine and proliferation was monitored by incorporation of ³ H-thymidine.

«· ··» and · 9 9 * «· • · · • · • ♦ • • · · • ·

Polyclonal stimulation with concavalin A (25 mcg / ml) served as a positive control. The stimulation index was calculated by normalizing to autoreactivity, which in all cases was very close to the baseline incorporation (background). CTLA4-Ig or humanized 5c8 was added to MIR-1. day at a concentration of 100 mcg / ml to 0.01 mcg / ml. Combination therapy was performed with different concentrations of CTLA4-Ig. and a constant concentration of humanized 5c8 that was 50 mcg / ml.

Analysis of peripheral blood lymphocyte phenotypes was performed prior to transplantation and periodically thereafter and after termination of therapy. 0.2 ml of heparinized whole blood diluted in phosphate buffered saline with 1% calf fetal serum was evaluated in the tests. Monoclonal antibodies ΤΙΙ, ΒΙ labeled with FITC (Coulter) and FN18 (donated by Dr. David M. Neville, Jr) were used to evaluate the percentage of CD2 (T cells / NK cells), CD20 (B cells) and CD3 (T cells) positive cells. .). Red blood cells were removed from the preparation by ACK lysis buffer (0.15M NH ₄ Cl, 1.0 mM KHCO ₃ , 1.0M Na ₂ EDTA, pH 7.3) followed by staining. Immediately thereafter, cells were analyzed by flow cytometry using a FACSCAN (Becton Dickinson).

Kidney allografts

Allotransplantations of the kidneys were performed as previously described. Briefly, unrelated cynomolgus rhesus seronegative for SIV (simian immunodeficiency virus), simian retrovirus and herpes B virus were obtained from the Primate Center (University of Wisconsin) or LABS (Yemassee, SC). Surgery was performed under general anesthesia, achieved using ketamine (1 mg / kg, i.m.), xylazine (1 mg / kg, i.m.) and halolate (1%, by inhalation). Transplants were performed between genetically different individuals in donor-recipient pairs, which was

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determined by MHC analysis described above. Animals were heparinized during organ harvesting and implantation (100 units / kg). The allograft was implanted by standard microvascular technique, creating an end to side anastomosis between the donor renal artery and the recipient's distal aorta and the cava vein also by the donor's renal vein. Thus, a primary ureteroneocystostomy was created. Bilateral native nephrectomy was terminated prior to occlusion.

Pal animals were given intravenous fluids for 36 hours until oral intake was sufficient. Trometafrine sulfa was administered for 3 days as a surgical antibiotic prophylaxis. Each animal received 81 mg aspirin on the day of surgery. The need for analgesia was assessed very frequently and was maintained by butorphanol administered intramuscularly. Animals were weighed weekly. Skin stitches were removed after 7 to 10 days. CTLA4-Ig or 5c8 were administered in dosages and according to the dosing schedule based on experience gained. No other animals were administered. Serum creatinine and blood electrolytes (Na +, K +, Ca 2+) and hemoglobin were determined every other day until steady state was reached, and then weekly thereafter.

intravenously depending on the immunopharmaceutical

Pathological analyzes

Animals suspected of healing were biopsied using a 20-gauge needle sampling device (Biopty-Cut, Bard). Standard. hematoxylin and eosin staining was performed on frozen or formalin fixed tissue to confirm the diagnosis of rejection. The animals were then sacrificed when anuria or weight loss reached 15% of body weight before transplantation,

• In accordance with AAALAC standards. All animals were subjected to a complete overall examination at the time of death and. also histopathological examination.

Results

Both CTLA4-Ig and humanized 5c8 inhibited cynomolgus MLR in a dose-dependent manner. However, CTLA4-Ig was more effective than humanized 5c8 in preventing T cell proliferation when used as a single agent. A significant decrease in thymidine incorporation was seen in CTLA4-Ig at concentration. 0.1 mcg / ml and further inhibition was achieved at higher concentrations. A more modest reduction in proliferation was achieved by humanized 5c8 at a concentration of 0.01 mcg / ml, but at increasing concentrations no more inhibition was observed. When tested in combination, both agents inhibited proliferation about 100-fold more effectively than in single use alone. The dose-response study was repeated for 3 different naive animals with identical results. Thus, CTLA4-Ig and humanized 5c8 synergistically prevent allograft rejection in vivo.

Twelve kidney allotransplantations were performed. Four animals received the transplant without any immunological intervention. These animals rejected the graft on days 5, 7, 7 and 8. Histological examination of the kidneys revealed acute cell rejection characterized by diffuse interstitial and tubular infiltration of lymphocytes with edema and cell necrosis. One animal received a five-day course of CTLA4-Ig (10mg / kg / day) starting at the time of transplantation and the graft survival time was extended to 20 days. Loss. graft was due to cell rejection indistinguishable from that in control animals.

One animal was treated with a single dose of 20 mg / kg CTLA4-Ig on the day of transplant followed by a 12 day administration of 10 mg / kg every other day, thereby prolonging graft survival to 30 days.

Again, graft loss was due to acute cellular rejection. By extrapolation based on previously published rat model of heterotopic cardiac allograft, donor-specific lymphocyte transfusion (10 ⁸ ) from lymph nodes was provided at the time of transplantation to these two animals.

Two animals were treated with humanized 5c8 alone. Both animals received a dose of 20 mg / kg every other day starting on the day of surgery until the 14th day after surgery (a total of 8 doses). In both animals, prolonged graft survival without healing was evident, although transient increases in creatinine levels were noted during the second and third weeks after surgery. The healing occurred on day 95 and day 100 after surgery. It was performed on both animals. biopsy to confirm diagnosis. Both animals then received 7 doses of humanized 5c8 (20 mg / kg dose, one animal every other day and the other animal daily) and both had normal graft function without any visible adverse effects. The animals remained alive and in good health more than 150 days after transplantation.

Two animals received both CTLA4-Ig and 5c8 at a dose of 20 mg / kg. Each agent was administered every other day starting on the day of surgery and continued until day 14 after surgery. One animal had healed 32 days after surgery. The second was 100 days free of healing, but then healing occurred similarly to animals receiving 5c8 alone. Biopsy showed acute cellular rejection. The initial CTLA4-Ig regimen along with humanized 5c8 was repeated in this animal and creatinine levels returned to baseline (1.0). MLR analysis after this treatment showed a donor specific loss of reactivity. Reactivity to a third party was maintained. On day 165 post-transplant, the animal was sacrificed as required by protocol

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- 31 - _? weight loss. At that time graft function was normal. On autopsy, it was found that the animal had enterocolitis caused by Shigela and Camphylobacter, a general infection in macaques. This disease has occurred in many animals of the original colony, as well as in several untreated animals. No other pathological changes were found, specifically, no manifestation of lymphoproliferative disease or opportunistic infection was found. Histologically, it was found that the graft had isolated lymphocyte nests in the interstitium, but no evidence of tubular infiltration, glomerular damage, or parenchymal necrosis was found.

As with animals treated with 5c8 'alone, both animals experienced transient increases in creatinine with concomitant increases in graft volume during the fourth week after surgery. Probably this transplant swelling was a reflection of the second wave of lymphocyte infiltration and therefore led to a modification of the dosing schedule so that both compounds were administered on the day of surgery and then 2, 4, 6, 8, 12, 16 and 28 days after surgery.

Two animals were treated with this modified method. Both survived for more than 150 days without symptoms of healing, without disease or changes in renal function. After 100 days with no signs of healing, the MLR test against the donor cells and the third species was repeated. No changes in in vitro reactivity were observed. The same tests were repeated after 150 days with no signs of rejection and resulted in the same results. Both animals retained strong reactivity against the donor and third species cells, but did not heal the transplant from the donor. No animal showed toxicity in any treatment. No fever, anorexia, haemodynamic abnormalities, nor opportunistic infections were reported. The animals were kept under standard conditions and had free contact with other animals in the colony. She kept her normal

MM • • • • 444 • 44 • · · β ·

- 32 weight gains. Laboratory biochemical tests and haematological parameters such as hemoglobin levels or white blood cell counts remained normal. The percentages of cells expressing CD, 2, CD3, and CD20 were unaffected by any treatment regimen used. Specifically, none of the animals experienced a decrease in the number of T cells either during or after treatment.

Example 2 '

Further preclinical studies in model primate renal allograft system

The primate renal allograft model system described above was further used to test various additional and / or further improved therapeutic regimens based on the humanized monoclonal antibody 5c8 either in monotherapy or combined with other therapeutic agents such as CTLA4-Ig, MMF, tacrolimus, corticosteroids or combinations thereof.

Monotherapy of renal allograft in primates

Two animals were given monotherapy with the following induction and maintenance regimen: Induction, the schedule included the administration of a dose of 20 mg / kg 5c8 on days -1, 0, 3, 10 and 18, with day 0 being the day of kidney transplant surgery. The maintenance phase consisted of monthly doses of 20 mg / kg 5c8 starting on day 28. The treated animals were free of healing as assessed by monitoring lymphocyte subpopulations and / or serum creatinine levels on days 163 and 170.

• 44

4 ·

4 4 • 4 4 4

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Two additional animals were given 5c8 monotherapy with a standard induction and maintenance regimen: The induction schedule included the administration of 20 mg / kg of 5c8 on days -1, 0, 3, 10 and 18, with day 0 being the day of kidney transplant surgery. The maintenance phase consisted of monthly doses of 10 mg / kg 5c8 beginning on day 28. The treated animals were substantially healed on day 148 and day 148 of the study.

Two other animals were also given 5c8 monotherapy with a low-dose induction regimen and a low-dose maintenance regimen: The induction schedule included a dose of 10 mg / kg 5c8 on days -1, 0, 3, 10 and 18, with day 0 being the day of performance kidney transplant surgery. The maintenance phase consisted of monthly doses of 10 mg / kg 5c8 beginning on day 28. The treated animals were essentially healed on days 38 and 9 of the study.

Two other animals received 5c8 monotherapy with an even lower dose induction regimen and an even lower dose maintenance regimen: The induction schedule included a 5 mg / kg dose of 5c8 on days -1, 0, 3, 10, and 18, with day 0 being day of kidney transplant surgery. The maintenance phase consisted of monthly doses of 10 mg / kg 5c8 starting on day 28. The treated animals healed the graft on days 7-10.

Combination therapy of renal allograft in primates

All animals received 5c8 therapy. a standard induction phase with a dose of 20 mg / kg and a maintenance regimen also with a dose of 20 mg / kg as described above, in combination with other immunosuppressive regimens:

Three animals were treated with a combination therapy containing corticosteroids (methylprednisolone, 5 day induction phase) and mycophenol mofetil, (MMF, 20 mg / kg poBID) in therapeutically.

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- 34 effective doses. The treated animals were essentially free of healing at 143, 81 and 80 days of the study. In contrast, control animals treated with similar doses of MMF and corticosteroids but without 5c8 therapy healed the implanted kidney on study day 7.

Two additional animals were treated with combination therapy containing the immunosuppressant tacrolimus (formerly referred to as FK506) at therapeutically effective doses (1.5 to 2 mg / kg poBID, administered 10ng / ml). The treated animals were essentially free of healing at days 31 and 36 of the study.

Two additional animals were treated with combination therapy containing CTLA4-Ig at therapeutically effective doses. The treated animals were essentially free of healing at 122 and 3 days of the study. '

Conclusions based on preclinical model studies

All of the above results demonstrate that induction of graft integration by a CD40 binding agent, CD154, humanized 5c8 alone alone, results in long-term survival of tissue allograft. The effect of humanized 5c8 is synergistically combined with the effect of CD28 signaling disrupter, CTLA4-Ig, and is compatible with some known immunosuppressants and / or immunomodulators.

Equivalence

The invention may be practiced in other specific forms without departing from the spirit of the invention. All of these embodiments are, therefore, to be construed as merely illustrative and not limiting of the invention. The subject matter of the invention is defined by the following claims, and all changes that are equivalent in meaning and scope are also within the scope of the invention.

Claims (20)

, 1. Use of a CD40: CD154 Binding Agent for the manufacture of a medicament for inhibiting rejection of a tissue graft by a recipient, a primate graft. Use according to claim 1, wherein the binding agent CD40: CD154 is an anti-CD40L compound (anti-CD154). The use of claim 2, wherein the anti-CD40L compound is a monoclonal antibody. The use of claim 3, wherein the monoclonal antibody binds to a 5c8 antigen. The use of claim 4, wherein the monoclonal antibody has antigen-specific binding characteristics of antibody 5c8 prepared from ATCC No. HB 10916. 6. Use of a CD40: CD154 Binding Agent for the manufacture of a medicament for reversing the acute rejection of transplanted tissue in a primate graft recipient. The use of claim 6, wherein the transplanted tissue is selected from kidney, liver, cardiac, pancreatic, cutaneous, vascular, nerve and bone and cartilage tissue. Use of a CD40: CD154 binding canceling agent for the manufacture of a medicament for prolonging the survival of transplanted tissue in a primate graft recipient. Use of a CD40: CD154 binding canceling agent for the manufacture of a medicament for alleviating immunological complications in transplant tissue failure in a primate graft recipient. Use according to claim 1, 6. 8 or 9, wherein the transplanted tissue is an allogene for a primate recipient, or wherein transplantovaná primate. tissue is xenogenic for the recipient who is 11.Use according to claims 1, 6, 8 or 9, where transplanted tissue suspended cells. composed of isolated or 12.Use according to claim 11, where are isolated or suspended cells selected from the group consisting of: (a) peripheral blood cells; and (b) bone marrow cells or any hematopoietic component thereof. The use of claim 1, 6, 8 or 9, comprising the additional step of administering to the primate an effective amount of an immunosuppressive or immunomodulatory agent. The use of claim 13, wherein the immunosuppressive or immunomodulatory agent is an agent that disrupts costimulatory signaling of T cells by CD28, or an agent that disrupts calcineurin signaling. The use of claim 14, wherein the immunosuppressive or immunomodulatory agent is selected from the group consisting of: a corticosteroid, an antiproliferative agent, cyclosporin, 37,999 · • · · 999 9 98 99 • · · 9 9 · 9 9 • ··· 999 4 4 49 99 tacrolimus, sirolimus, mycophenolate mofetil, mizorubin, deoxyspergualin, brequinar sodium, leflunomide and azaspiran. Use of a CD4O: CD154 binding agent for the manufacture of a medicament for inhibiting graft rejection by a primate recipient, wherein an effective amount of said medicament is administered to the primate on the 2nd, 4th, 6th, 8th, 12th, 16th and 28th. day, counted from the day of graft implantation to the primate. Use of a CD40: CD154 Binding Agent for the manufacture of a medicament for inhibiting graft rejection by a primate recipient, wherein an effective amount of said medicament is administered to a potential graft recipient, and one day thereafter, the graft is implanted to the primate and simultaneously administered an effective amount of said medicament is administered to the primate on days 3, 10, 18, and 28, calculated from the date of implantation. The use of claim 17, wherein the medicament is administered repeatedly monthly, starting one month after the 28th day from the date of implantation. Use of a CD40: CD154 Binding Agent for the manufacture of a medicament for reversing the acute rejection of a transplanted tissue in a primate recipient graft, wherein said medicament is administered to a primate on the day it exhibits symptoms of acute graft rejection and is then administered 3. , Days 10, 18 and 28. Use according to claim 19, wherein the medicament is administered to the primate repeatedly monthly, starting one month after the 28th day from the day of discovery of the symptoms of acute graft rejection. • 99 • • 99 • 9 9 9 • ·! 9 9 999 9 · 999 99 999 21.Application according to claim 1, 6, 8, 9, 16 17 or 19, where primate is human. 22.Application according to claim 1, 6, 8, 9, 16 17 or 19, where agent CD40 debonding: CD154 is administered graft recipients - to a primate in a manner selected from the group consisting of: a) parenteral administration, (b) biocompatible or bioerodible. a sustained release implant, c) infusion pump implant, (d) oral or enteral administration; and e) topical administration. The use of claim 1, 6, 8, 9, 20, 21 or 23, wherein the CD40: CD154 binding debonding agent is administered to the graft donor or to the transplanted graft prior to integration of the graft into a primate recipient graft. 24. A composition comprising: a monoclonal antibody having the antigen - specific characteristics of antibody 5c8 prepared from ATCC No. HB 10916, or an antigen binding fragment thereof, and immunosuppressive or. an immunomodulatory compound selected from the group consisting of: a) an agent that disrupts T cell costimulation by CD28, b) a calcineurin signaling disrupter, c) a corticosteroid or antiproliferative agent, d) tacrolimus, sirolimus, mycophenolate mofetil, mizorubin, deoxyspergualin, azquinarpine sodium, brequinunan sodium, brequinunan sodium, brequinunan sodium; .