**6.3 Antibodies**

Antibodies are sometimes used as a quick and potent immunosuppressive therapy to prevent acute rejection episodes; additionally, antibodies such as monoclonal anti-CD20 antibodies are used in targeted treatment of lymphoproliferative or autoimmune disorders.

Antibodies interact with lymphocyte surface antigens, resulting in the depletion of circulating thymus-derived lymphocytes and interference with cell-mediated and humoral immune responses. Lymphocyte depletion occurs either by complement-dependent lysis in the intravascular spaces or by opsonization and subsequent macrophage phagocytosis. Adverse effects such as fever, chills, thrombocytopenia, leukopenia, and headache typically occur with the first few doses.

Heterologous polyclonal antibodies are obtained from the serum of animals (e.g., rabbit, horse) that were injected with the patient's thymocytes or lymphocytes. Currently, antilymphocyte (ALG) and anti-thymocyte antigens (ATG) are used. They are part of the

epithelial adhesion; emigration; chemotaxis; phagocytosis; respiratory burst; and the release of various inflammatory mediators including, but not limited to, lysosomal enzymes, cytokines, tissue plasminogen activator, and chemokines from neutrophils, macrophages,

Cytostatics inhibit cell division. In immunotherapy, they are used in smaller doses than when used to treatment malignant diseases. They affect both T and B cell proliferation. As

The alkylating agents used in immunotherapy include cyclophosphamide (nitrogen mustards), nitrosoureas, platinum compounds, and others. Cyclophosphamide is probably the most potent immunosuppressive compound. In small doses, it is very efficient for the treatment of systemic lupus erythematosus, autoimmune hemolytic anemias, Wegener's granulomatosis, and other immune diseases. However, high doses cause pancytopenia and

Methotrexate is a folic acid analogue. It binds dihydrofolate reductase and prevents the synthesis of tetrahydrofolate. In addition to its use in transplant patients, it is used for the

It is extensively used to control transplant rejection reactions. Azathioprine is nonenzymatically cleaved to mercaptopurine, which acts as a purine analogue and an inhibitor of DNA synthesis. Additionally, mercaptopurine itself can also be administered

By preventing the clonal expansion of lymphocytes in the induction phase of the immune response, it affects both the cell and the humoral immunity. It is also efficient in the

Antibodies are sometimes used as a quick and potent immunosuppressive therapy to prevent acute rejection episodes; additionally, antibodies such as monoclonal anti-CD20 antibodies are used in targeted treatment of lymphoproliferative or autoimmune disorders. Antibodies interact with lymphocyte surface antigens, resulting in the depletion of circulating thymus-derived lymphocytes and interference with cell-mediated and humoral immune responses. Lymphocyte depletion occurs either by complement-dependent lysis in the intravascular spaces or by opsonization and subsequent macrophage phagocytosis. Adverse effects such as fever, chills, thrombocytopenia, leukopenia, and headache typically

Heterologous polyclonal antibodies are obtained from the serum of animals (e.g., rabbit, horse) that were injected with the patient's thymocytes or lymphocytes. Currently, antilymphocyte (ALG) and anti-thymocyte antigens (ATG) are used. They are part of the

treatment of autoimmune diseases such as rheumatoid arthritis and Behcet's disease.

they are the most effective, purine analogs are most frequently administered.

and mast cells.

**6.2 Cytostatics** 

*Alkylating agents* 

hemorrhagic cystitis.

treatment of autoimmune diseases.

occur with the first few doses.

*Antimetabolites* 

directly.

**6.3 Antibodies** 

treatment strategy for cases of steroid-resistant acute rejection reaction and grave aplastic anemia. However, they are added primarily to other immunosuppressives to diminish their dosage and toxicity. They also enable transition to cyclosporine therapy.

Polyclonal antibodies inhibit T cells and cause their depletion through either complementmediated cytolysis or cell-mediated opsonization that is followed by macrophage phagocytosis and degradation. In this way, polyclonal antibodies inhibit cell-mediated immune reactions, including graft rejection, delayed hypersensitivity, and graft-versus-host disease (GVHD); additionally they also influence thymus-dependent antibody production.

In 2005, two preparations became available: Atgam, obtained from horse serum, and Thymoglobulin, obtained from rabbit serum. Polyclonal antibodies affect all lymphocytes and cause general immunosuppression, possibly leading to post-transplant lymphoproliferative disorders (PTLD) or serious infections, such as those caused by cytomegalovirus. To reduce these risks, treatment is provided in a hospital, where adequate isolation from infection is available. Atgam and Thymoglobulin are usually administered for five days intravenously in the appropriate quantity. Patients stay in the hospital as long as three weeks to give the immune system time to recover to a point where there is no longer a risk of serum sickness.

Monoclonal antibodies are directed towards precisely defined antigens. Therefore, they cause fewer side effects. Especially significant are the antibodies directed against IL-2 receptor- (CD25-) and CD3. They are used to prevent organ transplant rejection, but also to track changes in the lymphocyte subpopulations. It is reasonable to expect the development of similar new drugs in the future.

#### *T-cell receptor directed antibodies*

Muromonab-CD3 is a murine anti-CD3 IgG2a monoclonal antibody that prevents T-cell activation and proliferation by binding the T-cell receptor complex that is present on all mature T cells. As such, it is one of the most potent immunosuppressive substances; it is commonly administered to control episodes of steroid- and/or polyclonal antibody-resistant acute rejection. Because it acts more specifically than polyclonal antibodies, it is also used prophylactically in transplantations.

However,the mechanism of action of muromonab-CD3 is only partially understood. It is known that the molecule binds the TCR/CD3 receptor complex. In the first few administrations, this binding non-specifically activates T-cells, leading to a serious syndrome 30 to 60 minutes later that is characterized by fever, myalgia, headache, and arthralgia. Sometimes the syndrome develops into a life-threatening reaction of the cardiovascular system and the central nervous system that requires lengthy therapy for recovery. After this period, CD3 blocks TCR-antigen binding and causes either conformational changes or the removal of the entire TCR3/CD3 complex from the T-cell surface. This lowers the number of available T-cells, perhaps by sensitizing them for uptake by tissue-resident macrophages. Also, cross-linking of CD3 molecules activates an intracellular signal that causes T cell anergy or apoptosis unless the cells receive another signal through a co-stimulatory molecule. Additionally, anti-CD3 antibodies shift the balance from Th1 to Th2 cells.

However, patients can develop neutralizing antibodies that reduce the effectiveness of muromonab-CD3. Muromonab-CD3 can also cause excessive immunosuppression. Although anti-CD3 antibodies act more specifically than polyclonal antibodies, they lower the cellmediated immunity significantly, predisposing patients to opportunistic infections and malignancies.

#### *IL-2 receptor directed antibodies*

Interleukin-2 is an important immune system regulator that is necessary for the clonal expansion and survival of activated T lymphocytes. Its effects are mediated by the trimeric cell surface IL-2 receptor, which is comprised of α, β, and γ chains. IL-2 receptor α (CD25, Tcell activation antigen, TAC) is expressed only by activated T lymphocytes. Therefore, it is of special significance for selective immunosuppressive treatment, and research has been focused on the development of effective and safe anti-IL-2 antibodies. Through the use of recombinant gene technology, murine anti-TAC antibodies have been modified, leading to the introduction of two chimeric mouse/human anti-Tac antibodies in 1998: basiliximab (Simulect) and daclizumab (Zenapax). These drugs act by binding the IL-2 receptor α chain, preventing IL-2-induced clonal expansion of activated lymphocytes and shortening their survival. Basiliximab and daclizumab are currently being used for the prevention of acute organ rejection after bilateral kidney transplantation; they are similarly effective and are associated with minimal side effects.

#### *Other monoclonal antibodies*

Efalizumab is a humanized monoclonal antibody that targets the lymphocyte functionassociated antigen-1 (LFA-1) receptor through the CD11a side chain. Efalizumab (Raptiva), a drug indicated for psoriasis, was withdrawn from the US market on June 8, 2009 because of potential risks for progressive multifocal leukoencephalopathy (PML). PML is a rapidly progressive infection of the central nervous system caused by the JC virus that leads to death or severe disability. Demyelination associated with PML results from the JC virus infection. JC virus belongs to the genus *Polyomavirus* of the *Papovaviridae* family. PML should be considered in any patient exhibiting new-onset neurologic manifestations who has taken efalizumab. For more information, please see the Food and Drug Administration MedWatch Safety Alert.

Monoclonal antibodies against B7-1 (CD80) and B7-2 (CD86) have been developed to block T-cell CD28 activation and proliferation. In a recent trial, one of these antibodies, belatacept, was not inferior to cyclosporine as a means of preventing acute rejection after renal transplantation. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) can simultaneously inhibit B7-1 and B7-2 interactions with CD28 and has been used successfully in animal models, demonstrating a beneficial effect on chronic allograft rejection. Monoclonal anti-CD45-RB, leflunomide, FK778, FTY720, alemtuzumab (anti-CD52 antibody), and rituximab are some of the other agents that are currently in different phases of evaluation. Other antibodies targeting CD28 are also in development.

#### **6.4 Immunophilin-binding agents**

The available immunophilin-binding agents are cyclosporine and tacrolimus. These agents are calcineurin inhibitors that primarily suppress T lymphocyte activation by inhibiting IL-2 production. They are associated with numerous toxicities that are often dose-dependent. Nephrotoxicity occurs with both the drugs. Hirsutism, gingival hypertrophy, hypertension, and hyperlipidemia develop more often with cyclosporine than tacrolimus.

Calcineurin inhibitors and azathioprine have been associated with post-transplant malignancies and skin cancers in organ transplant recipients. The results of several studies suggest that calcineurin inhibitors have oncogenic properties that are predominantly linked to the production of cytokines that promote tumor growth, metastasis, and angiogenesis. This drug has been reported to reduce the frequency of regulatory T cells; additionally, after converting from CNI monotherapy to a mycophenolate monotherapy, patients were found to have increased graft success and T-Reg frequencies.

#### *Cyclosporine*

106 Liver Transplantation – Basic Issues

anti-CD3 antibodies act more specifically than polyclonal antibodies, they lower the cellmediated immunity significantly, predisposing patients to opportunistic infections and

Interleukin-2 is an important immune system regulator that is necessary for the clonal expansion and survival of activated T lymphocytes. Its effects are mediated by the trimeric cell surface IL-2 receptor, which is comprised of α, β, and γ chains. IL-2 receptor α (CD25, Tcell activation antigen, TAC) is expressed only by activated T lymphocytes. Therefore, it is of special significance for selective immunosuppressive treatment, and research has been focused on the development of effective and safe anti-IL-2 antibodies. Through the use of recombinant gene technology, murine anti-TAC antibodies have been modified, leading to the introduction of two chimeric mouse/human anti-Tac antibodies in 1998: basiliximab (Simulect) and daclizumab (Zenapax). These drugs act by binding the IL-2 receptor α chain, preventing IL-2-induced clonal expansion of activated lymphocytes and shortening their survival. Basiliximab and daclizumab are currently being used for the prevention of acute organ rejection after bilateral kidney transplantation; they are similarly effective and are

Efalizumab is a humanized monoclonal antibody that targets the lymphocyte functionassociated antigen-1 (LFA-1) receptor through the CD11a side chain. Efalizumab (Raptiva), a drug indicated for psoriasis, was withdrawn from the US market on June 8, 2009 because of potential risks for progressive multifocal leukoencephalopathy (PML). PML is a rapidly progressive infection of the central nervous system caused by the JC virus that leads to death or severe disability. Demyelination associated with PML results from the JC virus infection. JC virus belongs to the genus *Polyomavirus* of the *Papovaviridae* family. PML should be considered in any patient exhibiting new-onset neurologic manifestations who has taken efalizumab. For more information, please see the Food and Drug Administration MedWatch

Monoclonal antibodies against B7-1 (CD80) and B7-2 (CD86) have been developed to block T-cell CD28 activation and proliferation. In a recent trial, one of these antibodies, belatacept, was not inferior to cyclosporine as a means of preventing acute rejection after renal transplantation. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) can simultaneously inhibit B7-1 and B7-2 interactions with CD28 and has been used successfully in animal models, demonstrating a beneficial effect on chronic allograft rejection. Monoclonal anti-CD45-RB, leflunomide, FK778, FTY720, alemtuzumab (anti-CD52 antibody), and rituximab are some of the other agents that are currently in different phases

The available immunophilin-binding agents are cyclosporine and tacrolimus. These agents are calcineurin inhibitors that primarily suppress T lymphocyte activation by inhibiting IL-2 production. They are associated with numerous toxicities that are often dose-dependent. Nephrotoxicity occurs with both the drugs. Hirsutism, gingival hypertrophy, hypertension,

of evaluation. Other antibodies targeting CD28 are also in development.

and hyperlipidemia develop more often with cyclosporine than tacrolimus.

malignancies.

*IL-2 receptor directed antibodies* 

associated with minimal side effects.

**6.4 Immunophilin-binding agents** 

*Other monoclonal antibodies* 

Safety Alert.

Since its introduction in 1983, Cyclosporine has become one of the most widely used immunosuppressive drugs. It is a cyclic fungal peptide that is comprised of 11 amino acids. Cyclosporine is thought to bind to the cytosolic protein cyclophilin (an immunophilin) of immunocompetent T lymphocytes. The complex of Cyclosporine and cyclophilin inhibits the phosphatase calcineurin. The drug also inhibits lymphokine production and cytokine release, leading to the reduced functionality of effector T-cells. The adverse events of cyclosporine would be hair growth as well as trembling and shaking of hands. For cyclosporine, the target conventional trough levels (C0) levels were adapted as 300–400 ng/mL during the rst postoperative month, 100–200 ng/mL for up to 1 year, and approximately 100 ng/mL or less thereafter. Cyclosporine is used to treat acute rejection reactions, but has been increasingly been replaced by newer, less nephrotoxic immunosuppressants.

#### *Tacrolimus*

Tacrolimus (trade name Prograf) is a product of the bacterium *Streptomyces tsukubaensis*. It is a macrolide lactone that acts by inhibiting calcineurin. Although tacrolimus is used particularly for liver and kidney transplants, in some clinics it is used for heart, lung and heart/lung transplants. It binds to the immunophilin FKBP1A; the tacrolimus-FKBP1A complex then binds to calcineurin and inhibits its phosphatase activity. In this way, Tacrolimus prevents the cell from transitioning from the G0 into G1 phase of the cell cycle. While tacrolimus binds to a different intracellular protein (FKBP-12) compared to cyclosporine, it has the same mechanism of action of cyclosporine, is more potent than Cyclosporine and is associated with less pronounced side effects compared to cyclosporine. Tacrolimus treatment was started orally to maintain a C0 whole blood level of 10–15 ng/mL initially with reduction of the dose to obtain C0 levels between 3-10 ng/mL at 1 year after liver transplant. However, neurotoxicity, alopecia, and posttransplant diabetes mellitus develop more frequently with tacrolimus compared to cyclosporine.

#### *Advagraf*

Advagraf is a new oral formulation of tacrolimus with prolonged-release characteristics compared to the currently authorised product Prograf(t). Advagraf therapy requires careful monitoring by adequately qualified and equipped personnel. Because the later product is nationally authorised, the invented name may vary depending on the country of authorisation. Advagraf is the first calcineurin inhibitor formulated to enable once daily oral dosing and it is expected that it may help to improve compliance with dosing and cause less interference with the daily life activities of the patient. This medicinal product should only be prescribed, and changes in immunosuppressive therapy initiated, by physicians experienced in immunosuppressive therapy and the management of transplant patients.

#### *Voclosporin*

Voclosporin is a calcineurin inhibitor that is under development by Isotechnika as of 2010. This company uses calcineurin as a surrogate marker to assess the amount of immunosuppression achieved using drugs in this category. Other companies also have next generation drugs in this class in their pipelines.
