Section X - Drugs Used for Immunomodulation doc

Section X. Drugs Used for Immunomodulation
Chapter 53. Immunomodulators: Immunosuppressive Agents,
Tolerogens, and Immunostimulants
Overview
This chapter provides a brief overview of the immune response as background for understanding the
mechanism of action of immunomodulatory agents. The general principles of pharmacological
immunosuppression are discussed in the context of potential targets, major indications, and
unwanted side effects. Four major classes of immunosuppressive drugs are discussed:
glucocorticoids (see also Chapter 60: Adrenocorticotropic Hormone; Adrenocortical Steroids and
Their Synthetic Analogs; Inhibitors of the Synthesis and Actions of Adrenocortical Hormones),
calcineurin inhibitors, antiproliferative and antimetabolic agents (see also Chapter 52:
Antineoplastic Agents), and antibodies. The "holy grail" of immunomodulation is the induction and
maintenance of immune tolerance, the active state of antigen-specific nonresponsiveness.
Approaches expected to overcome the risks of infections and tumors with immunosuppression are
reviewed. These include costimulatory blockade, donor-cell chimerism, soluble human leukocyte
antigens (HLA), and antigen-based therapies. Lastly, a general discussion of the limited number of
immunostimulant agents is presented, concluding with an overview of active and passive
immunization. New immunotherapeutic approaches will address not only the issues of specific drug
toxicities and efficacy but also long-term economic, metabolic, and quality-of-life outcomes.
The Immune Response
The immune system evolved to discriminate self from nonself. Multicellular organisms were faced
with the problem of destroying infectious invaders (microbes) or dysregulated self (tumors) while
leaving normal cells intact. These organisms responded by developing a robust array of receptor-
mediated sensing and effector mechanisms broadly described as innate and adaptive. Innate, or
natural, immunity is primitive, does not require priming, is of relatively low affinity, but is broadly
reactive. Adaptive, or learned, immunity is antigen-specific, depends upon antigen exposure or
priming, and can be of very high affinity. The two arms of immunity work closely together, with the
innate immune system being most active early in an immune response and adaptive immunity
becoming progressively dominant over time. The major effectors of innate immunity are
complement, granulocytes, monocytes/macrophages, natural killer cells, mast cells, and basophils.
The major effectors of adaptive immunity are B and T cells. B cells make antibodies; T cells

function as helper, cytolytic, and regulatory (suppressor) cells. These cells are important in the
normal immune response to infection and tumors but also mediate transplant rejection and
autoimmunity (Janeway et al. , 1999; Paul, 1999). Immunoglobulins (antibodies) on the B-cell
surface are receptors for a large variety of specific structural conformations. In contrast, T cells
recognize antigens as peptide fragments in the context of self major histocompatibility complex
(MHC) antigens (called HLA in human beings) on the surface of antigen-presenting cells (APCs),
such as dendritic cells, macrophages, and other cell types expressing MHC class I (HLA-A, B, and
C) and class II antigens (HLA-DR, DP, and DQ) in human beings. Once activated by specific
antigen recognition via their respective clonally restricted cell-surface receptors, both B and T cells
are triggered to differentiate and divide, leading to release of soluble mediators (cytokines,
lymphokines) that perform as effectors and regulators of the immune response.
The impact of the immune system in human disease is enormous. Developing vaccines against
emerging infectious agents from human immunodeficiency virus (HIV) to Ebola virus is among the
most critical challenges facing the research community. Immune system-mediated diseases are
significant health-care problems. Immunological diseases are growing at epidemic proportions that
require aggressive and innovative approaches to the development of new treatments. These diseases
include a broad spectrum of autoimmune diseases such as rheumatoid arthritis, diabetes mellitus,
systemic lupus erythematosus, and multiple sclerosis; solid tumors and hematologic malignancies;
infectious diseases; asthma; and various allergic conditions. Furthermore, one of the great
therapeutic opportunities for the treatment of many disorders is organ transplantation. However,
immune system–mediated graft rejection remains the single greatest barrier to widespread use of
this technology. An improved understanding of the immune system has led to the development of
new therapies to treat immune system–mediated diseases. This chapter briefly reviews drugs used to
modulate the immune response in three ways: immunosuppression, tolerance, and
immunostimulation.
Immunosuppression
Immunosuppressive drugs are used to dampen the immune response in organ transplantation and
autoimmune disease. In transplantation, the major classes of drugs used today are: (1)
glucocorticoids, (2) calcineurin inhibitors, and (3) antiproliferative/antimetabolic agents. These
drugs have met with a high degree of clinical success in treating conditions such as acute immune

rejection of organ transplants and severe autoimmune diseases. However, such therapies require
lifelong use and nonspecifically suppress the entire immune system, exposing patients to
considerably higher risks of infection and cancer. The calcineurin inhibitors and steroids, in
particular, are nephrotoxic and diabetogenic, thus limiting their usefulness in a variety of clinical
settings.
Monoclonal and polyclonal antibody preparations directed at reactive T cells are important adjunct
therapies and provide a unique opportunity to selectively target specific immune-reactive cells and
thus promote more specific treatments. Finally, new agents recently have expanded the arsenal of
immunosuppressive agents. In particular, sirolimus and anti–CD25 [interleukin (IL)-2 receptor]
antibodies (basiliximab, daclizumab) are being used to target growth factor pathways, substantially
limiting clonal expansion and thus promoting tolerance. The most commonly used
immunosuppressive drugs are described below. Nevertheless, many new, more selective,
therapeutic agents are on the horizon and are expected to revolutionize immunotherapy in the next
decade.
General Approach to Organ Transplantation Therapy
Organ transplant therapy is organized around five general principles. The first principle is careful
patient preparation and selection of the best available ABO-compatible HLA match for organ
donation (Legendre and Guttman, 1989). Second, a multitiered approach to immunosuppressive
drug therapy, similar to that in cancer chemotherapy, is employed. Several agents are used
simultaneously, each of which is directed at a different molecular target within the allograft
response (Table 53–1; Krensky, et al. , 1990; Hong and Kahan, 2000a). Synergistic effects are
obtained through application of the various agents at relatively low doses, thereby limiting specific
toxicities while maximizing the immunosuppressive effect. The third principle is that greater
immunosuppression is required to gain early engraftment and/or to treat established rejection than
to maintain immunosuppression in the long term. Therefore, intensive induction and lower-dose
maintenance drug protocols are employed. Fourth, careful investigation of each episode of
transplant dysfunction is required, including evaluation for rejection, drug toxicity, and infection,
keeping in mind that these various problems can and often do coexist. The fifth principle involves
reduction or withdrawal of a therapeutic agent when its toxicity exceeds its benefit.
Sequential Immunotherapy

In many organ transplant centers, muromonab-CD3, anti-CD25 monoclonal antibodies, or
polyclonal antilymphocyte antibodies are used as induction therapy in the immediate
posttransplantation period (Wilde and Goa, 1996; Brennan et al. , 1999). This treatment enables
initial engraftment without the use of high doses of nephrotoxic calcineurin inhibitors. Such
protocols reduce the incidence of early rejection and appear to be particularly beneficial for patients
at high risk for graft rejection (broadly presensitized or retransplant patients, pediatric recipients, or
African Americans).
Maintenance Immunotherapy
The basic immunosuppressive protocol used in most transplant centers involves the use of multiple
drugs simultaneously. Therapy typically involves a calcineurin inhibitor, steroids, and
mycophenolate mofetil (a purine metabolism inhibitor), each directed at a discrete site in T-cell
activation (Suthanthiran et al. , 1996; Perico and Remuzzi, 1997). Glucocorticoids, azathioprine,
cyclosporine, tacrolimus, mycophenolate mofetil, sirolimus, and various monoclonal and polyclonal
antibodies currently are approved by the United States Food and Drug Administration (FDA) for
use in transplantation.
Therapy for Established Rejection
Although low doses of prednisone, calcineurin inhibitors, purine-metabolism inhibitors, or
sirolimus are effective in preventing acute cellular rejection, they are not as effective in blocking T
cells that already are activated, and they are not very effective against established, acute rejection or
for the total prevention of chronic rejection (Monaco et al. , 1999). Therefore, treatment of
established rejection requires the use of agents directed against activated T cells. These include
glucocorticoids in high doses (pulse therapy), polyclonal antilymphocyte antibodies, or
muromonab-CD3 monoclonal antibody.
Adrenocortical Steroids
The introduction of glucocorticoids as immunosuppressive drugs in the 1960s played a key role in
making organ transplantation possible. The chemistry, pharmacokinetics, and drug interactions of
adrenocortical steroids are described in Chapter 60: Adrenocorticotropic Hormone; Adrenocortical
Steroids and Their Synthetic Analogs; Inhibitors of the Synthesis and Actions of Adrenocortical
Hormones. Prednisone, prednisolone, and other glucocorticoids are used alone and in combination
with other immunosuppressive agents for treatment of transplant rejection and autoimmune

disorders.
Mechanism of Action
The immunosuppressive effects of glucocorticoids long have been known, but the specific
mechanism(s) of their immunosuppressive action remains somewhat elusive (Rugstad, 1988; Beato,
1989). Steroids lyse and possibly induce the redistribution of lymphocytes, causing a rapid,
transient decrease in peripheral blood lymphocyte counts. To effect longer-term responses, steroids
bind to receptors inside cells, and either these receptors or glucocorticoid-induced proteins bind to
DNA in the vicinity of response elements that regulate the transcription of numerous other genes
(see Chapter 60: Adrenocorticotropic Hormone; Adrenocortical Steroids and Their Synthetic
Analogs; Inhibitors of the Synthesis and Actions of Adrenocortical Hormones). Additionally,
glucocorticoid-receptor complexes increase I B expression, thereby curtailing activation of NF B,
which results in increased apoptosis of activated cells (Auphan et al. , 1995). Of central importance
in this regard is the downregulation of important proinflammatory cytokines, such as IL-1 and IL-6.
T cells are inhibited from making IL-2 and proliferating. The activation of cytotoxic T lymphocytes
is inhibited. Neutrophils and monocytes display poor chemotaxis and decreased lysosomal enzyme
release. Therefore, glucocorticoids have broad antiinflammatory effects on cellular immunity. In
contrast, they have relatively little effect on humoral immunity.
Therapeutic Uses
Glucocorticoids commonly are used in combination with other immunosuppressive agents to both
prevent and treat transplant rejection. High doses of intravenous methylprednisolone sodium
succinate (SOLU-MEDROL, A-METHAPRED) (pulses) are used to reverse acute transplant rejection
and acute exacerbations of selected autoimmune disorders (Shinn et al. , 1999; Laan et al. , 1999).
There are numerous indications for glucocorticoids (Zoorob and Cender, 1998). They are
efficacious for treatment of graft-versus-host disease in bone-marrow transplantation. Among
autoimmune disorders, glucocorticoids are used routinely to treat rheumatoid and other arthritides,
systemic lupus erythematosus, systemic dermatomyositis, psoriasis and other skin conditions,
asthma and other allergic disorders, inflammatory bowel disease, inflammatory ophthalmic
diseases, autoimmune hematologic disorders, and acute exacerbations of multiple sclerosis. In
addition, glucocorticoids limit allergic reactions that occur with other immunosuppressive agents
and are used in transplant recipients to block first-dose cytokine storm caused by treatment with

muromonad-CD3 (see below).
Toxicity
Unfortunately, because there are numerous steroid-responsive tissues and genes, the extensive use
of steroids has resulted in disabling and life-threatening adverse effects in many patients. These
effects include growth retardation, avascular necrosis of bone, osteopenia, increased risk of
infection, poor wound healing, cataracts, hyperglycemia, and hypertension (see Chapter 60:
Adrenocorticotropic Hormone; Adrenocortical Steroids and Their Synthetic Analogs; Inhibitors of
the Synthesis and Actions of Adrenocortical Hormones). The advent of concomitant
glucocorticoid/cyclosporine regimens has allowed a reduction in the dosages of steroids
administered, yet steroid-induced morbidity is still a major problem in many transplant patients.
Calcineurin Inhibitors
Perhaps the most effective immunosuppressive drugs in routine clinical use are calcineurin
inhibitors, cyclosporine and tacrolimus, drugs that target intracellular signaling pathways induced
as a consequence of T-cell-receptor activation (Schreiber and Crabtree, 1992). Although they are
structurally unrelated (Figure 53–1) and bind to different (but related) molecular targets, the
mechanisms of action of cyclosporine and tacrolimus in inhibiting normal T-cell signal transduction
are the same (Figure 53–2). Cyclosporine and tacrolimus do not act per se as immunosuppressive
agents. Instead, these drugs "gain function" after binding to cyclophilin or FKBP-12, resulting in
subsequent interaction with calcineurin to block the activity of this phosphatase. Calcineurin-
catalyzed dephosphorylation is required for movement of a component of the nuclear factor of
activated T lymphocytes (NFAT) into the nucleus (Figure 53–2). NFAT, in turn, is required for
induction of a number of cytokine genes, including that for interleukin-2 (IL-2), a prototypic T-cell
growth and differentiation factor.

Figure 53–1. Chemical Structures of Immunosuppressive Drugs: Azathioprine,
Mycophenolate Mofetil, Cyclosporine, Tacrolimus, and Sirolimus.

Figure 53–2. Mechanisms of Action of Cyclosporine, Tacrolimus, and
Sirolimus. Both cyclosporine and tacrolimus bind to immunophilins [cyclophilin
and FK506-binding protein (FKBP), respectively], forming a complex that binds

the phosphatase calcineurin and inhibits the calcineurin-catalyzed
dephosphorylation essential to permit movement of the nuclear factor of activated
T cells (NFAT) into the nucleus. NFAT is required for transcription of
interleukin-2 (IL-2) and other growth and differentiation–associated cytokines
(lymphokines). Sirolimus (rapamycin) works at a later stage in T-cell activation,
downstream of the IL-2 receptor. Sirolimus also binds FKBP, but the FKBP-
sirolimus complex binds to and inhibits the mammalian target of rapamycin
(mTOR), a kinase involved in cell-cycle progression (proliferation). DG,
diacylglycerol; PIP
2
, phosphatidylinositol bisphosphate; PLC, phospholipase C;
PKC, protein kinase C; TCR, T-cell receptor. (From Pattison et al. , 1997, with
permission.)
Cyclosporine
Chemistry
Cyclosporine (cyclosporin A) is a cyclic polypeptide consisting of 11 amino acids, produced as a
metabolite of the fungus species Beauveria nivea (Borel et al. , 1976). Of note, all amide nitrogens
are either hydrogen bonded or methylated, the single D-amino acid is at position 8, the methyl
amide between residues 9 and 10 is in the cis configuration, and all other methyl amide moieties are
in the trans form (Figure 53–1). Since cyclosporine is lipophilic and highly hydrophobic, it must be
solubilized for clinical administration.
Mechanism of Action
Cyclosporine suppresses some humoral immunity but is more effective against T cell–dependent
immune mechanisms such as those underlying transplant rejection and some forms of autoimmunity
(Kahan, 1989). It preferentially inhibits antigen-triggered signal transduction in T lymphocytes,
blunting expression of many lymphokines, including IL-2, as well as expression of antiapoptotic
proteins. Cyclosporine forms a complex with cyclophilin, a cytoplasmic receptor protein present in
target cells. This complex binds to calcineurin, inhibiting Ca
2+
-stimulated dephosphorylation of the

cytosolic component of NFAT (Schreiber and Crabtree, 1992). When the cytoplasmic component of
NFAT is dephosphorylated, it translocates to the nucleus, where it complexes with nuclear
components required for complete T-cell activation, including transactivation of IL-2 and other
lymphokine genes. Calcineurin enzymatic activity is inhibited following physical interaction with
the cyclosporine/cyclophilin complex. This results in the blockade of NFAT dephosphorylation;
thus, the cytoplasmic component of NFAT does not enter the nucleus, gene transcription is not
activated, and the T lymphocyte fails to respond to specific antigenic stimulation. Cyclosporine also
increases expression of transforming growth factor (TGF- ), a potent inhibitor of IL-2-stimulated
T-cell proliferation and generation of cytotoxic T lymphocytes (CTL) (Khanna et al. , 1994).
Disposition and Pharmacokinetics
Cyclosporine can be administered intravenously or orally. The intravenous preparation
(SANDIMMUNE Injection) is provided as a solution in an ethanol-polyoxyethylated castor oil vehicle
which must be further diluted in 0.9%sodium chloride solution or 5%dextrose solution before
injection. The oral dosage forms include soft gelatin capsules and oral solutions. Cyclosporine
supplied in the original soft gelatin capsule is absorbed slowly with 20% to 50% bioavailability. A
modified microemulsion formulation (NEORAL) was developed to improve absorption and was
approved by the FDA for use in the United States in 1995 (Noble and Markham, 1995). It has more
uniform and slightly increased bioavailability compared to SANDIMMUNE and is provided as 25-mg
and 100-mg soft gelatin capsules and a 100-mg/ml oral solution. Since SANDIMMUNE and NEORAL
are not bioequivalent, they cannot be used interchangeably without supervision by a physician and
monitoring of drug concentration in plasma. Comparison of blood concentrations in published
literature and in clinical practice must be performed with a detailed knowledge of the assay system
employed. Although generic cyclosporine formulations have become available (Halloran, 1997), the
most carefully studied generic product recently was withdrawn from the United States market by
the FDA because of questions raised about bioequivalence.
As described above, absorption of cyclosporine is incomplete following oral administration. The
extent of absorption depends upon several variables, including the individual patient and
formulation used. The elimination of cyclosporine from the blood is generally biphasic, with a
terminal half-life of 5 to 18 hours (Faulds et al. , 1993; Noble and Markham, 1995). After
intravenous infusion, clearance is approximately 5 to 7 ml/min per kg in adult recipients of renal

transplants, but results differ by age and patient populations. For example, clearance is slower in
cardiac transplant patients and more rapid in children. The relationship between administered dose
and the area under the plasma concentration–versus-time curve (AUC; see Chapter 1:
Pharmacokinetics: The Dynamics of Drug Absorption, Distribution, and Elimination) is linear
within the therapeutic range, but the intersubject variability is so large that individual monitoring is
required (Faulds et al. , 1993; Noble and Markham, 1995).
Following oral administration of cyclosporine (as NEORAL), the time to peak blood concentrations
is 1.5 to 2.0 hours (Faulds et al. , 1993; Noble and Markham, 1995). Administration with food both
delays and decreases absorption. High- and low-fat meals consumed within 30 minutes of
administration decrease the AUC by approximately 13% and the maximum concentration by 33%.
This makes it imperative to individualize dosage regimens for outpatients.
Cyclosporine is distributed extensively outside the vascular compartment. After intravenous dosing,
the steady-state volume of distribution has been reported to be as high as 3 to 5 liters/kg in solid-
organ transplant recipients.
Only 0.1% of cyclosporine is excreted unchanged in urine (Faulds et al. , 1993). Cyclosporine is
extensively metabolized in the liver by the cytochrome-P450 3A (CYP3A) enzyme system and to a
lesser degree by the gastrointestinal tract and kidneys (Fahr, 1993). At least 25 metabolites have
been identified in human bile, feces, blood, and urine (Christians and Sewing, 1993). Although the
cyclic peptide structure of cyclosporine is relatively resistant to metabolism, the side chains are
extensively metabolized. All of the metabolites have both reduced biological activity and toxicity
compared to the parent drug. Cyclosporine and its metabolites are excreted principally through the
bile into the feces, with only approximately 6% being excreted in the urine. Cyclosporine also is
excreted in human milk. In the presence of hepatic dysfunction, dosage adjustments are required.
No adjustments generally are necessary for dialysis or renal failure patients.
Therapeutic Uses
Clinical indications for cyclosporine are kidney, liver, heart, and other organ transplantation;
rheumatoid arthritis; and psoriasis (Faulds et al. , 1993). Its use in dermatology is discussed in
Chapter 65: Dermatological Pharmacology. Cyclosporine generally is recognized as the agent that
ushered in the modern era of organ transplantation, increasing the rates of early engraftment,
extending graft survival for kidneys, and making cardiac and liver transplantation possible.

Cyclosporine usually is used in combination with other agents, especially glucocorticoids and either
azathioprine or mycophenolate mofetil and, most recently, sirolimus. The dosage of cyclosporine
used is quite variable, depending upon the organ transplanted and the other drugs used in the
specific treatment protocol(s). The initial dose generally is not given pretransplant because of the
concern about neurotoxicity. Especially for renal transplant patients, therapeutic algorithms have
been developed to delay cyclosporine introduction until a threshold renal function has been attained.
The amount of the initial dose and reduction to maintenance dosing is sufficiently variable that no
specific recommendation is provided here. Dosage is guided by signs of rejection (too low a dose),
renal or other toxicity (too high a dose), and close monitoring of blood levels. Great care must be
taken to differentiate renal toxicity from rejection in kidney transplant patients. Because adverse
reactions have been ascribed frequently to the intravenous formulation, this route of administration
is discontinued as soon as the patient is able to take an oral form of the drug.
In rheumatoid arthritis, cyclosporine is used in cases of severe disease that have not responded to
methotrexate. Cyclosporine can be used in combination with methotrexate, but the levels of both
drugs must be monitored closely (Baraldo et al. , 1999). In psoriasis, cyclosporine is indicated for
treatment of adult nonimmunocompromised patients with severe and disabling disease who have
failed other systemic therapies (Linden and Weinstein, 1999). Because of its mechanism of action,
there is a theoretical basis for the use of cyclosporine in a variety of other T cell–mediated diseases
(Faulds et al. , 1993). Cyclosporine has been reported to be effective in Behçet's acute ocular
syndrome, endogenous uveitis, atopic dermatitis, inflammatory bowel disease, and nephrotic
syndrome when standard therapies have failed.
Toxicity
The principal adverse reactions to cyclosporine therapy are renal dysfunction, tremor, hirsutism,
hypertension, hyperlipidemia, and gum hyperplasia (Burke et al. , 1994). Nephrotoxicity is limiting
and occurs in the majority of patients treated. It is the major indication for cessation or modification
of therapy. Hypertension may occur in approximately 50% of renal transplant and almost all cardiac
transplant patients. Combined use of calcineurin inhibitors and glucocorticoids is particularly
diabetogenic, with diabetes being more frequent in patients treated with tacrolimus than in those
receiving cyclosporine.
Drug Interactions

Cyclosporine interacts with a wide variety of commonly used drugs, and close attention must be
paid to drug interactions. Any drug that affects microsomal enzymes, especially the CYP3A system,
may affect cyclosporine blood concentrations (Faulds et al. , 1993). Substances that inhibit this
enzyme can decrease cyclosporine metabolism and increase blood concentrations. These include
calcium channel blockers (e.g., verapamil, nicardipine), antifungal agents (e.g., fluconazole,
ketoconazole), antibiotics (e.g., erythromycin), glucocorticoids (e.g., methylprednisolone), HIV-
protease inhibitors (e.g., indinavir), and other drugs (e.g., allopurinol and metoclopramide). In
addition, grapefruit and grapefruit juice block the CYP3A system and increase cyclosporine blood
concentrations and thus should be avoided by patients receiving the drug. In contrast, drugs that
induce CYP3A activity can increase cyclosporine metabolism and decrease blood concentrations.
Drugs that can decrease cyclosporine concentrations in this manner include antibiotics (e.g.,
nafcillin and rifampin), anticonvulsants (e.g., phenobarbital, phenytoin), and other drugs (e.g.,
octreotide, ticlopidine). In general, close monitoring of cyclosporine blood levels and the levels of
other drugs is required when such combinations are used.
Interactions between cyclosporine and sirolimus have led to the recommendation that
administration of the two drugs be separated by time. Sirolimus aggravates cyclosporine-induced
renal dysfunction, while cyclosporine increases sirolimus-induced hyperlipemia and
myelosuppression. Other cyclosporine–drug interactions of concern include additive nephrotoxicity
when coadministered with nonsteroidal antiinflammatory drugs and other drugs that cause renal
dysfunction; elevation in methotrexate levels when the two drugs are coadministered; and reduced
clearance of other drugs, including prednisolone, digoxin, and lovastatin.
Tacrolimus
Tacrolimus (PROGRAF, FK506) is a macrolide antibiotic produced by Streptomyces tsukubaensis
(Goto et al. , 1987). Its formula is shown in Figure 53–1.
Mechanism of Action
Like cyclosporine, tacrolimus inhibits T-cell activation by inhibiting calcineurin (Schreiber and
Crabtree, 1992). Tacrolimus binds to an intracellular protein, FK506-binding protein–12 (FKBP-
12), an immunophilin structurally related to cyclophilin. A complex of tacrolimus-FKBP-12,
calcium, calmodulin, and calcineurin then forms, and calcineurin phosphatase activity is inhibited.
As described for cyclosporine and depicted in Figure 53–2, the inhibition of phosphatase activity

prevents dephosphorylation and nuclear translocation of NFAT and leads to inhibition of T-cell
activation. Thus, although the intracellular receptors differ, cyclosporine and tacrolimus appear to
share a single common pathway for immunosuppression (Plosker and Foster, 2000).
Disposition and Pharmacokinetics
Tacrolimus is available for oral administration as capsules (0.5, 1, and 5 mg) and a sterile solution
for injection (5 mg/ml). Immunosuppressive activity resides primarily in the parent drug. Because
of intersubject variability in pharmacokinetics, individualization of dosing is required for optimal
therapy (Fung and Starzl, 1995). Whole blood, rather than plasma, is the most appropriate sampling
compartment to describe tacrolimus pharmacokinetics. Gastrointestinal absorption is incomplete
and variable. Food decreases both the rate and extent of absorption. Plasma protein binding of
tacrolimus is 75% to 99%, involving primarily albumin and
1
-acid glycoprotein. Its half-life is
about 12 hours. Tacrolimus is extensively metabolized in the liver by CYP3A, and at least some of
the metabolites are active. The bulk of excretion of parent drug and metabolites is in the feces. Less
than 1% of administered tacrolimus is excreted unchanged in the urine.
Therapeutic Uses
Tacrolimus is indicated for the prophylaxis of solid-organ allograft rejection in a manner similar to
cyclosporine and as rescue therapy in patients with rejection episodes despite "therapeutic" levels of
cyclosporine (Mayer et al. , 1997; The U.S. Multicenter FK506 Liver Study Group, 1994). The
recommended starting dose for tacrolimus injection is 0.03 to 0.05 mg/kg per day as a continuous
infusion. Recommended initial oral doses are 0.2 mg/kg per day for adult kidney transplant patients,
0.1 to 0.15 mg/kg per day for adult liver transplant patients, and 0.15 to 0.2 mg/kg per day for
pediatric liver transplant patients in two divided doses 12 hours apart. These dosages are intended to
achieve typical blood trough levels in the 5- to 20-ng/ml range. Pediatric patients generally require
higher doses than do adults (Shapiro, 1998).
Toxicity
Nephrotoxicity, neurotoxicity (tremor, headache, motor disturbances, seizures), gastrointestinal
complaints, hypertension, hyperkalemia, hyperglycemia, and diabetes are associated with
tacrolimus use (Plosker and Foster, 2000). As with cyclosporine, nephrotoxicity is limiting

(Mihatsch et al. , 1998; Henry, 1999). Tacrolimus has a negative effect on the pancreatic islet beta
cell, and both glucose intolerance and diabetes mellitus are well- recognized complications of
tacrolimus-based immunosuppression among adult solid-organ transplant recipients. As with other
immunosuppressive agents, there is an increased risk of secondary tumors and opportunistic
infections.
Drug Interactions
Because of its potential for nephrotoxicity, blood levels of tacrolimus and renal function should be
monitored closely, especially when tacrolimus is used with other potentially nephrotoxic drugs.
Coadministration with cyclosporine results in additive or synergistic nephrotoxicity; therefore, a
delay of at least 24 hours is required when switching a patient from cyclosporine to tacrolimus.
Since tacrolimus is metabolized mainly by CYP3A, the potential interactions described for
cyclosporine (above) apply for tacrolimus as well (Venkataramanan et al. , 1995; Yoshimura et al. ,
1999).
Antiproliferative and Antimetabolic Drugs
Sirolimus
Sirolimus (rapamycin; RAPAMUNE) is a macrocyclic lactone produced by Streptomyces
hygroscopicus (Vezina, et al. , 1975). Its structure is shown in Figure 53–1.
Mechanism of Action
Sirolimus inhibits T-lymphocyte activation and proliferation downstream of the IL-2 and other T-
cell growth factor receptors (Figure 53–2) (Kuo et al. , 1992). Sirolimus, like cyclosporine and
tacrolimus, is a drug whose therapeutic action requires formation of a complex with the
immunophilin, FKBP-12. However, the sirolimus-FKBP-12 complex does not affect calcineurin
activity, but binds to and inhibits the mammalian kinase, target of rapamycin (mTOR), which is a
key enzyme in cell-cycle progression (Brown et al. , 1994). Inhibition of this kinase blocks cell
cycle progression at the G
1
S phase transition. In animal models, sirolimus not only inhibits
transplant rejection, graft-versus-host disease, and a variety of autoimmune diseases, but its effect
also lasts several months after discontinuing therapy, suggesting a tolerizing effect (see"Tolerance,"
below) (Groth et al. , 1999).

Disposition and Pharmacokinetics
Following oral administration, sirolimus is absorbed rapidly and reaches a peak blood concentration
within about 1 hour after a single dose in healthy subjects and within about 2 hours after multiple
oral doses in renal transplant patients (Napoli and Kahan, 1996; Zimmerman and Kahan, 1997).
Systemic availability is approximately 15%, and blood concentrations are proportional to dose
between 3 and 12 mg/m
2
. A high-fat meal decreases peak blood concentration by 34%; sirolimus
therefore should be taken consistently either with or without food, and blood levels should be
monitored closely. About 40% of sirolimus in plasma is bound to protein, especially albumin. The
drug partitions into formed elements of blood, with a blood-to-plasma ratio of 38 in renal transplant
patients. Sirolimus is extensively metabolized by CYP3A4 and is transported by P-glycoprotein.
Seven major metabolites have been identified in whole blood (Salm et al. , 1999). Metabolites also
are detectable in feces and urine, with the bulk of total excretion being in feces. Although some of
its metabolites are active, sirolimus per se is the major component in whole blood and contributes
greater than 90% of the immunosuppressive effect. The blood half-life after multiple dosing in
stable renal transplant patients is 62 hours (Napoli and Kahan, 1996; Zimmerman and Kahan,
1997). A loading dose of three times the maintenance dose will provide nearly steady-state
concentrations within one day in most patients.
Therapeutic Uses
Sirolimus is indicated for prophylaxis of organ transplant rejection in combination therapy with a
calcineurin inhibitor and glucocorticoids (Kahan et al. , 1999a). In patients experiencing or at high
risk for calcineurin inhibitor–associated nephrotoxicity, sirolimus has been used with
glucocorticoids and mycophenolate mofetil to avoid permanent renal damage. The initial dosage in
patients 13 years or older who weigh less than 40 kg should be adjusted based on body surface area
(1 mg/m
2
per day) with a loading dose of 3 mg/m
2
. Data regarding doses for pediatric and geriatric

patients are lacking at this time (Kahan, 1999). It is recommended that the maintenance dose be
reduced by approximately one-third in patients with hepatic impairment (Watson et al. , 1999).
Toxicity
The use of sirolimus in renal transplant patients is associated with a dose-dependent increase in
serum cholesterol and triglycerides that may require treatment (Murgia et al. , 1996). While
immunotherapy with sirolimus per se is not nephrotoxic, patients treated with cyclosporine plus
sirolimus have impaired renal function compared to patients treated with cyclosporine and either
azathioprine or placebo. Renal function therefore must be monitored closely in such patients.
Lymphocoele, a known surgical complication associated with renal transplantation, has occurred
significantly more often in a dose-dependent fashion in sirolimus-treated patients, requiring close
postoperative follow-up. Other adverse effects include anemia, leukopenia, thrombocytopenia
(Hong and Kahan, 2000b), hypokalemia or hyperkalemia, fever, and gastrointestinal effects. As
with other immunosuppressive agents, there is an increased risk of neoplasms, especially
lymphomas, and infections. Prophylaxis for Pneumocystis carinii pneumonia and cytomegalovirus
is recommended (Groth et al. , 1999).
Drug Interactions
Since sirolimus is a substrate for cytochrome CYP3A4 and is transported by P-glycoprotein, close
attention to interactions with other drugs that are metabolized or transported by these proteins is
required (Yoshimura et al. , 1999). As noted above, cyclosporine and sirolimus interact, and their
administration should be separated by time. Dose adjustment may be required with coadministration
of sirolimus with cyclosporine, diltiazem, or rifampin. No dosage adjustment appears to be required
when sirolimus is coadministered with acyclovir, digoxin, glyburide, nifedipine, norgestrel/ethinyl
estadiol, prednisolone, or sulfamethoxazole/trimethoprim. This list is incomplete, and blood levels
and potential drug interactions must be monitored closely.
Azathioprine
Azathioprine (IMURAN) is a purine antimetabolite (Elion, 1993). It is an imidazolyl derivative of 6-
mercaptopurine (Figure 53–1).
Mechanism of Action
Following exposure to nucleophiles, such as glutathione, azathioprine is cleaved to 6-
mercaptopurine, which, in turn, is converted to additional metabolites that inhibit de novo purine

synthesis (Bertino, 1973). 6-Thio-IMP, a fraudulent nucleotide, is converted to 6-thio-GMP and
finally to 6-thio-GTP, which is incorporated into DNA and gene translation is inhibited (Chan et al. ,
1987). Cell proliferation is prevented, inhibiting a variety of lymphocyte functions. Azathioprine
appears to be a more potent immunosuppressive agent than does 6-mercaptopurine itself, which
may reflect differences in drug uptake or pharmacokinetic differences in the resulting metabolites.
Disposition and Pharmacokinetics
Azathioprine is well absorbed orally and reaches maximum blood levels within 1 to 2 hours after
administration. The half-life of azathioprine itself is about 10 minutes, and that of mercaptopurine is
about an hour. Other metabolites have half-lives of up to 5 hours. Blood levels have little predictive
value because of extensive metabolism, significant activity of many different metabolites, and high
tissue levels attained. Azathioprine and mercaptopurine are moderately bound to plasma proteins
and are partially dialyzable. Both azathioprine and mercaptopurine are rapidly removed from the
blood by oxidation or methylation in the liver and/or erythrocytes. Renal clearance is of little impact
in biological effectiveness or toxicity, but dose reduction is practiced in patients with renal failure.
Therapeutic Uses
Azathioprine was first introduced as an immunosuppressive agent in 1961, helping to make
allogeneic kidney transplantation possible (Murray et al. , 1963). It is indicated as an adjunct for
prevention of organ transplant rejection and in severe rheumatoid arthritis (Hong and Kahan, 2000a;
Gaffney and Scott, 1998). Although the dose of azathioprine required to prevent organ rejection and
minimize toxicity varies among patients, 3 to 5 mg/kg per day is the usual starting dose. Lower
initial doses (1 mg/kg per day) are used in treating rheumatoid arthritis. Complete blood count and
liver function tests should be monitored.
Toxicity
The major side effect of azathioprine is bone marrow suppression with leukopenia (common),
thrombocytopenia (less common), and/or anemia (uncommon). Other important adverse effects
include increased susceptibility to infections (especially varicella and herpes simplex viruses),
hepatotoxicity, alopecia, gastrointestinal toxicity, pancreatitis, and increased risk of neoplasia.
Drug Interactions
Xanthine oxidase, an enzyme of major importance in the catabolism of metabolites of azathioprine,
is blocked by allopurinol (Venkat Raman, et al. , 1990). If azathioprine and allopurinol are used in

the same patient, the azathioprine dose must be decreased to 25% to 33% of the usual dose, but it is
best not to use these two drugs together. Adverse effects resulting from coadministration of
azathioprine with other myelosuppressive agents or angiotensin converting enzyme inhibitors
include leukopenia, thrombocytopenia, and/or anemia as a result of myelosuppression.
Mycophenolate Mofetil
Mycophenolate mofetil (CELLCEPT) is the 2-morpholinoethyl ester of mycophenolic acid (MPA)
(Allison and Eugui, 1993). Its structure is shown in Figure 53–1.
Mechanism of Action
Mycophenolate mofetil is a prodrug that is rapidly hydrolyzed to the active drug, mycophenolic
acid (MPA), a selective, uncompetitive and reversible inhibitor of inosine monophosphate
dehydrogenase (IMPDH) (Natsumeda and Carr, 1993), an important enzyme in the de novo
pathway of guanine nucleotide synthesis. B and T lymphocytes are highly dependent on this
pathway for cell proliferation, while other cell types can use salvage pathways; MPA therefore
selectively inhibits lymphocyte proliferation and functions, including antibody formation, cellular
adhesion, and migration. The effects of MPA on lymphocytes can be reversed by adding guanosine
or deoxyguanosine to the cells.
Disposition and Pharmacokinetics
Mycophenolate mofetil undergoes rapid and complete metabolism to MPA after oral or intravenous
administration. MPA, in turn, is metabolized to the inactive phenolic glucuronide, MPAG. The
parent drug is cleared from the blood within a few minutes. The half-life of MPA is about 16 hours.
Negligible amounts (<1%) of MPA are excreted in the urine (Bardsley-Elliot et al. , 1999). Most
(87%) is excreted in the urine as MPAG. Plasma concentrations of both MPA and MPAG are
increased in patients with renal insufficiency. In early renal transplant patients (<40 days
posttransplant), plasma concentrations of MPA after a single dose of mycophenolate mofetil are
about half of those found in healthy volunteers or stable renal transplant patients. Studies in the
pediatric population are limited; safety and effectiveness in this population have not been
established (Butani et al. , 1999).
Therapeutic Uses
Mycophenolate mofetil is indicated for prophylaxis of transplant rejection and is typically used in
combination with glucocorticoids and a calcineurin inhibitor, but not with azathioprine (Kimball et

al. , 1995; Ahsan et al. , 1999; Kreis et al., 2000). Combined treatment with sirolimus is possible,
although potential drug interactions necessitate careful monitoring of drug levels. For renal
transplants, 1 g is administered orally or intravenously (over 2 hours) twice per day (2 g per day). A
higher dose, 1.5 g twice per day (3 g per day), is recommended for African-American renal
transplant patients and all cardiac transplant patients. Use of mycophenolate mofetil in other clinical
settings is under investigation.
Toxicity
The principal toxicities of mycophenolate mofetil are gastrointestinal and hematologic (Fulton and
Markham, 1996; Bardsley-Elliot et al. , 1999). These include leukopenia, diarrhea, and vomiting.
There also is an increased incidence of some infections, especially sepsis associated with
cytomegalovirus.
Drug Interactions
Potential drug interactions between mycophenolate mofetil and several other drugs commonly used
by transplant patients have been studied (Bardsley-Elliot et al. , 1999). There appear to be no
untoward effects produced by combination therapy with cyclosporine,
sulfamethoxazole/trimethoprim, or oral contraceptives. Mycophenolate mofetil has not been tested
with azathioprine. Coadministration with antacids containing aluminum or magnesium hydroxide
leads to decreased absorption of mycophenolate mofetil; thus, these drugs should not be
administered simultaneously. Mycophenolate mofetil should not be administered with
cholestyramine or other drugs that affect enterohepatic circulation. Such agents decrease plasma
MPA concentrations, probably by binding free MPA in the intestines. Acyclovir and gancyclovir
may compete with MPAG for tubular secretion, possibly resulting in increased concentrations of
both MPAG and the antiviral agents in the blood. This effect may be compounded in patients with
renal insufficiency.
Other Antiproliferative and Cytotoxic Agents
Many of the cytotoxic and antimetabolic agents used in cancer chemotherapy (see Chapter 52:
Antineoplastic Agents) are immunosuppressive due to their action on lymphocytes and other cells
of the immune system. Other cytotoxic drugs that have been used as immunosuppressive agents
include methotrexate, cyclophosphamide (CYTOXAN), thalidomide, and chlorambucil (LEUKERAN).
Methotrexate is used for treatment of graft-versus-host disease, rheumatoid arthritis, and psoriasis

as well as in anticancer thereapy (see Chapter 52: Antineoplastic Agents) (Grosflam and Weinblatt,
1991). Cyclophosphamide and chlorambucil are used in treating childhood nephrotic syndrome
(Neuhaus et al. , 1994) as well as in treating of a variety of malignancies (see Chapter 52:
Antineoplastic Agents). Cyclophosphamide also is widely used for treatment of severe systemic
lupus erythematosus (Valeri et al. , 1994). Leflunomide (ARAVA) is a pyrimidine-synthesis inhibitor
indicated for the treatment of adults with rheumatoid arthritis (Prakash and Jarvis, 1999). The drug
inhibits dihydroorotate dehydrogenase in the de novo pathway of pyrimidine synthesis. It is
hepatotoxic and can cause fetal injury when administered to pregnant women.
Antibodies
Both polyclonal and monoclonal antibodies against lymphocyte cell-surface antigens are widely
used for prevention and treatment of organ transplant rejection. Polyclonal antisera are generated by
repeated injections of human thymocytes (antithymocyte globulin, ATG) or lymphocytes
(antilymphocyte globulin, ALS) into animals such as horses, rabbits, sheep, or goats and then
purifying the serum immunoglobulin fraction (Mannick et al. , 1971). Although highly effective
immunosuppressive agents, these preparations vary in efficacy and toxicity from batch to batch.
The advent of hybridoma technology to produce monoclonal antibodies was a major advance in
immunology (Kohler and Milstein, 1975). It is now possible to make essentially unlimited amounts
of a single antibody of a defined specificity (Figure 53–3). These monoclonal reagents have
overcome the problems of variability in efficacy and toxicity seen with the polyclonal products, but
they are more limited in their target specificity. Thus, both polyclonal and monoclonal products
have a place in immunosuppressive therapy.

Figure 53–3. Generation of Monoclonal Antibodies. Mice are immunized with
the selected antigen, and spleen or lymph node is harvested and B cells separated.
These B cells are fused to a suitable B-cell myeloma that has been selected for its
inability to grow in medium supplemented with hypoxanthine, aminopterin, and
thymidine (HAT). Only myelomas that fuse with B cells can survive in HAT-
supplemented medium. The hybridomas expand in culture. Those of interest
based upon a specific screening technique are then selected and cloned by
limiting dilution. Monoclonal antibodies can be used directly as supernatants or

ascites fluid experimentally but are purified for clinical use. HPRT,
hypoxanthine-guanine phosphoribosyl transferase. (From Krensky, A.M., 1999,
with permission.)
Antithymocyte Globulin
Antithymocyte globulin (THYMOGLOBULIN) is a purified gamma globulin from the serum of rabbits
immunized with human thymocytes (Regan et al. , 1999). It is provided as a sterile, freeze-dried
product for intravenous administration after reconstitution with sterile water.
Mechanism of Action
Antithymocyte globulin contains cytotoxic antibodies that bind to CD2, CD3, CD4, CD8, CD11a,
CD18, CD25, CD44, CD45, and HLA class I and II molecules on the surface of human T
lymphocytes (Bourdage and Hamlin, 1995). The antibodies deplete circulating lymphocytes by
direct cytotoxicity (both complement and cell-mediated) and block lymphocyte function by binding
to cell surface molecules involved in the regulation of cell function.
Therapeutic Uses
Antithymocyte globulin is indicated for induction immunosuppression and the treatment of acute
renal transplant rejection in combination with other immunosuppressive agents (Mariat et al. ,
1998). Because it is a highly effective immunosuppressant, a course of antithymocyte-globulin
treatment often is given to renal transplant patients with delayed graft function to allow withdrawal
of nephrotoxic calcineurin inhibitors and thereby aid in recovery from ischemic reperfusion injury.
The recommended dose for acute rejection of renal grafts is 1.5 mg/kg per day (over 4 to 6 hours)
for 7 to 14 days. Mean T-cell counts fall by day 2 of therapy. It also is used for acute rejection of
other types of organ transplants and for prophylaxis of rejection (Wall, 1999). Studies to examine
its use as induction therapy at the time of transplantation are in progress (Szczech and Feldman,
1999).
Toxicity
The major side effects are fever and chills with the potential for hypotension. Premedication with
corticosteroids, acetaminophen, and/or an antihistamine and administration of the antiserum by
slow infusion (over 4 to 6 hours) into a large-diameter vessel minimize such reactions. Outright
serum sickness and glomerulonephritis can occur; anaphylaxis is a rare event. Hematologic
complications include leukopenia and thrombocytopenia. As with other immunosuppressive agents,

there is an increased risk of infection and malignancy, especially when multiple immunosuppressive
agents are used in combination. No drug interactions have been described, but anti-antibodies
develop, limiting repeated use of this or any other rabbit antibody preparations. As an example, in
one trial, 68% of patients developed antirabbit antibodies.
Monoclonal Antibodies
Anti-CD3 Monoclonal Antibodies
Antibodies directed at the CD3 antigen on the surface of human T lymphocytes have been used
since the early 1980s in human transplantation and have proven to be extremely effective
immunosuppressive agents. The original mouse IgG
2a
antihuman CD3 monoclonal antibody,
muromonab-CD3 (OKT3, ORTHOCLONE OKT
3
), is still used to reverse corticosteroid-resistant
rejection episodes (Cosimi, et al. , 1981).
Mechanism of Action
Muromonab-CD3 binds to CD3, a monomorphic component of the T-cell receptor complex
involved in antigen recognition, cell signaling, and proliferation (Hooks et al. , 1991). Antibody
treatment induces rapid internalization of the T-cell receptor, thereby preventing subsequent
recognition of antigen. Administration of the antibody is followed rapidly by depletion and
extravasation of a majority of T cells from the bloodstream and the peripheral lymphoid organs
such as lymph nodes and spleen. This absence of detectable T cells from the usual lymphoid regions
is secondary both to cell death following complement activation and activation-induced cell death
and to margination of T cells onto vascular endothelial walls and redistribution of T cells to
nonlymphoid organs such as the lungs. Muromonab-CD3 also induces a reduction in function of the
remaining T cells, as defined by lack of IL-2 production and great reduction in the production of
multiple cytokines, perhaps with the exception of IL-4 and IL-10.
Therapeutic Uses
Muromonab-CD3 is indicated for treatment of acute organ transplant rejection (Ortho Multicenter
Transplant Group, 1985; Woodle et al. , 1999; Rostaing et al. , 1999). Muromonab-CD3 is provided

as a sterile solution containing 5 mg per ampule. The recommended dose is 5 mg/day (in adults; less
for children) in a single intravenous bolus (less than one minute) for 10 to 14 days. Antibody levels
increase over the first three days and then level off. Circulating T cells disappear from the blood
within minutes of administration and return within approximately one week after termination of
therapy. Repeated use of muromonab-CD3 results in the immunization of the patient against the
mouse determinants of the antibody, which can neutralize and prevent its immunosuppressive
efficacy (Jaffers et al. , 1983). Thus, repeated treatment with the muromonab-CD3 or other mouse
monoclonal antibodies is contraindicated in many patients.
Toxicity
The major side effect of anti-CD3 therapy is the "cytokine release syndrome" (Wilde and Goa,
1996; Ortho Multicenter Transplant Study Group, 1985). Administration of glucocorticoids prior to
the injection of muromonab-CD3 prevents the release of cytokines and reduces first-dose reactions
considerably and is now a standard procedure. Antibody binding to the T-cell receptor complex
combined with Fc receptor (FcR)–mediated crosslinking is the basis for the initial activating
properties of this agent. The syndrome typically begins 30 minutes after infusion of the antibody
(but can occur later) and may persist for hours. The symptomatology usually is worst with the first
dose; both the frequency and severity decrease with subsequent doses. Common clinical
manifestations include high fever, chills/rigor, headache, tremor, nausea/vomiting, diarrhea,
abdominal pain, malaise, muscle/joint aches and pains, and generalized weakness. Less common
complaints include skin reactions and cardiorespiratory and central nervous system (CNS)
disorders, including aseptic meningitis. Potentially fatal, severe pulmonary edema, adult respiratory
distress syndrome, cardiovascular collapse, cardiac arrest, and arrhythmias have been described.
The syndrome is associated with and attributed to increased serum levels of cytokines [including
tumor necrosis factor (TNF)- , IL-2, IL-6, and interferon gamma], which are released by activated
T cells and/or monocytes. In several studies, the production of the TNF- cytokine has been shown
to be the major cause of the toxicity (Herbelin et al. , 1995). Fluid status of patients must be
monitored carefully before therapy; steroids and other premedications should be given, and a fully
competent resuscitation facility must be immediately available for patients receiving their first
several doses of this therapy. Other toxicities associated with anti-CD3 therapy include anaphylaxis
and the usual infections and neoplasms associated with immunosuppressive therapy. A high rate of

"rebound" rejection has been observed when muromonab-CD3 treatment is stopped (Wilde and
Goa, 1996).
New-Generation Anti-CD3 Antibodies
Recently, genetically altered anti-CD3 monoclonal antibodies have been developed that are
"humanized" to minimize the occurrence of anti-antibody responses and mutated to prevent binding
to FcRs (Friend et al. , 1999). The rationale for developing this new generation of anti-CD3
monoclonal antibodies is that they could induce selective immunomodulation in the absence of
toxicity associated with conventional anti-CD3 monoclonal antibody therapy. In initial clinical
trials, a humanized anti-CD3 monoclonal antibody that does not bind to FcRs reversed acute renal
allograft rejection in the absence of the first-dose cytokine-release syndrome (Woodle et al. , 1999).
Clinical efficacy of these agents in autoimmune diseases is being evaluated.
Anti-IL-2 Receptor (Anti-CD25) Antibodies
Daclizumab (ZENAPAX), a humanized murine complementarity- determining region (CDR)/human
IgG
1
chimeric monoclonal antibody, and basiliximab (SIMULECT), a murine-human chimeric
monoclonal antibody, have been produced by recombinant DNA technology (Wiseman and Faulds,
1999). The composite daclizumab antibody consists of human (90%) constant domains of IgG
1
and
variable framework regions of the Eu myeloma antibody and murine (10%) CDR of the anti-Tac
antibody.
Mechanism of Action
The antibodies bind with high affinity to the alpha subunit of the IL-2 receptor (p55 alpha, CD25)
present on the surface of activated, but not resting, T lymphocytes and block IL-2–mediated T-cell
activation events. Daclizumab has a somewhat lower affinity than does basiliximab.
Therapeutic Uses
Anti–IL-2-receptor monoclonal antibodies are recommended for prophylaxis of acute organ
rejection in adult patients as part of combination therapy (with glucocorticoids, a calcineurin
inhibitor, with or without azathioprine or mycophenolate mofetil) (Kovarik et al. , 1999; Hong and

Kahan, 1999; Kahan et al. , 1999b; Hirose et al., 2000). Daclizumab and basiliximab are supplied as
sterile concentrates that are diluted before intravenous administration. Renal transplant patients
receiving 1 mg/kg of daclizumab intravenously every 14 days for 5 doses have saturating blockade
of the IL-2 receptor for 120 days posttransplant (Vincenti et al. , 1998). No significant change in
circulating lymphocyte markers has been observed. Basiliximab is given for only two doses of 20
mg each, the first two hours before surgery and the second four days after.
Toxicity
No cytokine-release syndrome has been observed with these antibodies. Anaphylactic reactions can
occur. Although lymphoproliferative disorders and opportunistic infections may occur, as with
other immunosuppressive agents, the incidence ascribed to anti-CD25 treatment appears remarkably
low. No significant drug interactions with anti–IL-2-receptor antibodies have been described (Hong
and Kahan, 1999).
Infliximab
Infliximab (REMICADE) is a chimeric anti–TNF- monoclonal antibody containing a human
constant region and a murine variable region. It binds with high affinity to TNF- and prevents the
cytokine from binding to its receptors.
Patients with rheumatoid arthritis have elevated levels of TNF- in their joints, and patients with
Crohn's disease have elevated levels of TNF- in their stools. A clinical trial has revealed that
patients treated with infliximab plus methotrexate have fewer signs and symptoms of rheumatoid
arthritis than do patients treated with methotrexate alone. Patients with active Crohn's disease who
had not responded to other immunosuppressive therapies have shown improvement when treated
with infliximab, and patients with fistulizing Crohn's disease have had fewer draining fistulas after
treatment with the antibody. Infliximab is approved in the United States for treating the symptoms
of rheumatoid arthritis, in combination with methotrexate, in patients who do not respond to
methotrexate alone. Infliximab also is approved for use in the treatment of symptoms of moderately
to severely active Crohn's disease in patients who have failed to respond to conventional therapy
and in treatment to reduce the number of draining fistulas in Crohn's disease patients (see Chapter
39: Agents Used for Diarrhea, Constipation, and Inflammatory Bowel Disease; Agents Used for
Biliary and Pancreatic Disease). About 1 of 6 patients receiving infliximab has experienced an
infusion reaction within 1 to 2 hours after administration of the antibody. The reaction has included

fever, urticaria, hypotension, and dyspnea. Serious infections also have occurred in infliximab-
treated patients, most frequently in the upper respiratory and urinary tracts. The development of
antinuclear antibodies and, rarely, a lupus-like syndrome have been reported to occur after
treatment with infliximab.
A therapeutic agent related to infliximab in its mechanism of action, although not a monoclonal
antibody, is etanercept (ENBREL), which is a fusion protein containing the ligand-binding portion of
a human TNF- receptor linked to the Fc portion of human IgG
1
. Like infliximab, etanercept binds
to TNF- and prevents it from interacting with its receptors. It is approved in the United States for
treatment of the symptoms of rheumatoid arthritis in patients who have not responded to other
treatments. Etanercept can be used in combination with methotrexate in patients who have not
responded adequately to methotrexate alone. As with infliximab, serious infections have occurred
after treatment with etanercept. Injection-site reactions (erythema, itching, pain, or swelling) have
occurred in more than one-third of etanercept-treated patients.
Tolerance
Immunosuppression has concomitant risks of opportunistic infections and secondary tumors.
Therefore, the ultimate goal of research on organ transplantation and autoimmune diseases is the
induction and maintenance of immunologic tolerance, the active state of antigen-specific
nonresponsiveness (Krensky and Clayberger, 1994; Hackett and Dickler, 1999). If tolerance can be
attained, it would represent a true cure for conditions discussed above without the side effects of the
various immunosuppressive therapies discussed. The calcineurin inhibitors prevent tolerance
induction in some, but not all, preclinical models (Wood, 1991; Van Parijs and Abbas, 1998). In
contrast, in these same model systems, sirolimus does not prevent tolerance and, in fact, in some
cases promotes tolerance induction (Li et al. , 1998). Several other approaches have exciting
promise as well and are being evaluated in clinical trials. Because these approaches are still
experimental, they are only briefly discussed here.
Costimulatory Blockade
Induction of specific immune responses by T lymphocytes requires two signals: an antigen-specific
signal via the T-cell receptor and a costimulatory signal provided by molecules such as CD28 on the

T cell interacting with CD80 and CD86 on the antigen-presenting cell (APC) (Figure 53–4)
(Khoury et al. , 1999). Preclinical studies have shown that inhibition of the costimulatory signal can
induce tolerance (Larsen et al. , 1996; Kirk et al. , 1997). Experimental approaches to inhibition of
costimulation include a recombinant fusion protein molecule, CTLA4Ig, and anti-CD80 and/or anti-
CD86 monoclonal antibodies. CTLA4Ig contains the binding region of CTLA4, which is a CD28
homolog, and the constant region of the human IgG
1
. CTLA4Ig is a competitive inhibitor of CD28.
Both CTLA4Ig and a lytic anti-CD80 monoclonal antibody are in clinical trials. A second
costimulatory pathway undergoing clinical evaluation involves the interaction of CD40 on activated
T cells with CD40 ligand (CD154) on B cells, endothelium, and/or APCs (Figure 53–4). Among the
purported activities of anti-CD154 antibody treatment is its blockade of the induced B7 expression
following immune activation. At least two anti-CD154 monoclonal antibodies are under clinical
evaluation in organ transplantation and autoimmunity. Other antagonists of T-cell costimulation,
including anti-CD2, anti-ICAM-1 (CD54) and anti-LFA-1 monoclonal antibodies, have shown
promise in preclinical models of tolerance (Salmela et al. , 1999).

Figure 53–4. Costimulation. A. Two signals are required for T-cell activation.
Signal 1 is via the T-cell receptor (TCR) and signal 2 is via a costimulatory
receptor-ligand pair. Both signals are required for T-cell activation. Signal 1 in
the absence of signal 2 results in an inactivated T cell. B. One important
costimulatory pathway involves CD28 on the T cell and B7-1 (CD80) and B7-2
(CD86) on the antigen-presenting cell (APC). After a T cell is activated, it
expresses additional costimulatory molecules. CD152 is CD40 ligand, which
interacts with CD40 as a costimulatory pair. CD154 (CTLA4) interacts with
CD80 and CD86 to dampen or down-regulate an immune response. Antibodies
against CD80, CD86, and CD152 are being evaluated as potential therapeutic
agents. CTLA4-Ig, a chimeric protein consisting of part of an immunoglobulin
molecule and part of CD154, also has been tested as a therapeutic agent. (From
Clayberger et al., 2001, with permission.)

Donor Cell Chimerism
Another approach with exciting preliminary results is induction of chimerism (coexistence of cells
from two genetic lineages in a single individual) by any of a variety of protocols that first dampen
or eliminate immune function in the recipient with ionizing radiation, drugs such as
cyclophosphamide, and/or antibody treatment and then provide a new source of immune function
by adoptive transfer (transfusion) of bone marrow or hematopoietic stem cells (Starzl et al. , 1997;
Fuchimoto et al. , 1999; Spitzer et al. , 1999; Hale et al., 2000). Upon reconstitution of immune
function, the recipient no longer recognizes new antigens provided during a critical period as
"nonself." Such tolerance is long-lived and is less likely to be complicated by the use of calcineurin
inhibitors. Although the most promising approaches in this arena have been therapies that promote
the development of mixed or macrochimerism, in which substantial numbers of donor cells are
present in the circulation, some microchimerization approaches also have shown promise in the
development of long-term unresponsiveness.
Soluble HLA
In the precyclosporine era, blood transfusions were shown to be associated with improved outcomes
in renal transplant patients (Opelz and Terasaki, 1978). These findings gave rise to donor-specific
transfusion protocols that gave improved outcomes (Opelz et al. , 1997). After the introduction of
cyclosporine, however, these effects of blood transfusions disappeared, presumably due to the
efficacy of this drug in blocking T-cell activation. Nevertheless, the existence of a tolerance-
promoting effect of transfusions is irrefutable. It is possible that this effect is due to HLA molecules
on the surface of cells or in soluble forms. Recently, soluble HLA and peptides corresponding to
linear sequences of HLA molecules have been shown to induce immunologic tolerance in animal
models via a variety of mechanisms (Murphy and Krensky, 1999).
Antigens
Specific antigens provided in a variety of forms, but generally as peptides, induce immunologic
tolerance in preclinical models of diabetes, arthritis, and multiple sclerosis. Clinical trials of such
approaches are under way. The past decade has seen a revolution in our understanding of the basis
for immune tolerance. It is now well established that antigen/MHC complex binding to the T-cell
receptor/CD3 complex coupled with soluble and membrane-bound costimulatory signals initiates a
cascade of signaling events that lead to productive immunity. In addition, the immune response also

is regulated by a number of negative signaling events that control cell survival and expansion. For
the first time, in vitro and preclinical in vivo studies have demonstrated that one can selectively
inhibit immune responses to specific antigens without the associated toxicity of current
immunosuppressive therapies (Van Parijs and Abbas, 1998). With these new insights comes the
enormous promise of specific immune therapies to treat the vast array of immune disorders from
autoimmunity to transplant rejection. These new therapies will take advantage of a combination of
drugs that target the primary T-cell receptor–mediated signal either by blocking cell-surface
receptor interactions or inhibiting early signal transduction events. The drugs will be combined with
therapies that effectively block costimulation to prevent cell expansion and differentiation of those
cells that have engaged antigen while maintaining a noninflammatory milieu.
Immunostimulation
General Principles
In contrast to immunosuppressive agents that inhibit the immune response in transplant rejection
and autoimmunity, a few immunostimulatory drugs have been developed with applicability to
infection, immunodeficiency, and cancer. Major problems with such drugs are systemic
(generalized) effects at one extreme or limited efficacy at the other.
Immunostimulants
Levamisole
Levamisole (ERGAMISOL) was synthesized originally as an antihelminthic but appears to "restore"
depressed immune function of B cells, T cells, monocytes, and macrophages. Its only clinical
indication is as an adjuvant treatment with fluorouracil after surgical resection in patients with
Dukes' stage C colon cancer (Moertel et al. , 1990; Figueredo et al. , 1997). Its use has been
associated with sometimes fatal agranulocytosis.
Thalidomide
Thalidomide (THALOMID) is best known for the severe, life-threatening birth defects it has caused
when administered to pregnant women (Smithells and Newman, 1992; Lary et al. , 1999). For this
reason, it is available only under a restricted distribution program and can be prescribed only by
specially licensed physicians who understand the risk of teratogenicity if thalidomide is used during
pregnancy. Thalidomide should never be taken by women who are pregnant or who could
become pregnant while taking the drug. Nevertheless, it is indicated for the treatment of patients

with erythema nodosum leprosum (ENL) (Sampaio et al. , 1993). Its mechanism of action is unclear
(Tseng et al. , 1996). Reported immunologic effects vary substantially under different conditions.
For example, thalidomide has been reported to decrease circulating TNF- in patients with ENL but
to increase it in patients who are HIV-seropositive (Jacobson et al. , 1997). Alternatively, it has been
suggested that the drug affects angiogenesis (Miller and Stromland, 1999). The anti–TNF- effect
has led to its evaluation as a treatment for severe, refractory rheumatoid arthritis (Keesal et al. ,
1999).
Bacillus Calmette-Guérin (BCG)
Live BCG (TICE BCG, THERACYS) is an attenuated, live culture of the bacillus of Calmette and
Guérin strain of Mycobacterium bovis. BCG induces a granulomatous reaction at the site of
administration. This preparation is active against tumors by unclear mechanisms and is indicated for
treatment and prophylaxis of carcinoma in situ of the urinary bladder and for prophylaxis of
primary and recurrent stage Ta and/or T1 papillary tumors following transurethral resection
(Morales et al. , 1981; Paterson and Patel, 1998; Patard et al. , 1998). Adverse effects include
hypersensitivity, shock, chills, fever, malaise, and immune complex disease.
Recombinant Cytokines
Interferons
Although interferons (alpha, beta, and gamma) initially were identified by their antiviral activity,
these agents have important immunomodulatory activities as well (Johnson et al. , 1994; Tilg and
Kaser, 1999; Ransohoff, 1998). The interferons bind to specific cell-surface receptors that initiate a
series of intracellular events: induction of certain enzymes, inhibition of cell proliferation, and
enhancement of immune activities, including increased phagocytosis by macrophages and
augmentation of specific cytotoxicity by T lymphocytes (Tompkins, 1999). Recombinant interferon
alfa-2b (IFN-alpha 2, INTRON A) is obtained from Escherichia coli by genetic engineering. It is a
member of a family of naturally occurring small proteins with molecular weights of 15,000 to
27,600 daltons, produced and secreted by cells in response to viral infections and other inducers.
Interferon alfa-2b is indicated in the treatment of a variety of tumors, including hairy cell leukemia,
malignant melanoma, follicular lymphoma, and AIDS-related Kaposi's sarcoma (Punt, 1998;
Bukowski, 1999; Sinkovics and Horvath, 2000). It also is indicated for infectious diseases, chronic
hepatitis B, and condylomata acuminata. In addition, it is supplied in combination with ribavirin

(REBETRON) for treatment of chronic hepatitis C in patients with compensated liver function not
treated previously with interferon alfa-2b or who have relapsed following interferon alfa-2b therapy
(Lo Iacono et al., 2000). Flu-like symptoms, including fever, chills, and headache, are the most
common adverse effects seen after interferon alfa-2b administration. Adverse experiences involving
the cardiovascular system (hypotension, arrhythmias, and, rarely, cardiomyopathy and myocardial
infarction) and CNS (depression, confusion) are among other, less-frequent side effects.
Interferon gamma-1b (ACTIMMUNE) is a recombinant polypeptide that differs from other interferons
in causing activation of phagocytes and generation within them of oxygen metabolites that are toxic
to a number of microorganisms. It is indicated in reducing the frequency and severity of serious
infections associated with chronic granulomatous disease. Adverse reactions to it can include fever,
headache, rash, fatigue, gastrointestinal distress, anorexia, weight loss, myalgia, and depression.
Interferon beta-1a (AVONEX), a 166–amino acid recombinant glycoprotein, and interferon beta-1b
(BETASERON), a 165–amino acid recombinant protein, have antiviral and immunomodulatory
properties. They are FDA-approved for the treatment of relapsing and relapsing-remitting multiple
sclerosis to reduce the frequency of clinical exacerbations. The mechanism of their action in
multiple sclerosis is unclear. Flu-like symptoms (fever, chills, myalgia) and injection-site reactions
have been common adverse effects.
Further discussion of the use of these and other interferons in the treatment of viral diseases can be
found in Chapter 50: Antimicrobial Agents: Antiviral Agents (Nonretroviral).
Interleukin-2
Human recombinant interleukin-2 (aldesleukin, PROLEUKIN; des-alanyl-1, serine-125 human IL-2)
is produced by recombinant DNA technology in E. coli (Taniguchi and Minami, 1993). This
recombinant form differs from native IL-2 in that it is not glycosylated, has no amino terminal
alanine, and has a serine substituted for the cysteine at amino acid 125 (Doyle et al. , 1985). The
potency of the preparation is represented in International Units (IU) in a lymphocyte proliferation
assay such that 1.1 mg of recombinant IL-2 protein equals 18 million IU. Aldesleukin has the
following in vitro biologic activities of native IL-2: enhancement of lymphocyte proliferation and
growth of IL-2-dependent cell lines; enhancement of lymphocyte-mediated cytotoxicity and killer
cell activity; and induction of interferon-gamma activity (Winkelhake et al. , 1990; Whittington and
Faulds, 1993). In vivo administration of aldesleukin in animals produces multiple immunologic

effects in a dose-dependent manner. There is profound activation of cellular immunity with
lymphocytosis, eosinophilia, thrombocytopenia, and release of multiple cytokines (TNF, IL-1,
interferon gamma). Aldesleukin is indicated for the treatment of adults with metastatic renal cell
carcinoma and melanoma. Administration of aldesleukin has been associated with serious
cardiovascular toxicity resulting from capillary leak syndrome, which involves loss of vascular tone
and leak of plasma proteins and fluid into the extravascular space. Hypotension, reduced organ
perfusion, and death may occur. An increased risk of disseminated infection due to impaired
neutrophil function also has been associated with aldesleukin treatment.
Immunization
Immunization may be active or passive. Active immunization involves stimulation with an antigen
to develop immunologic defenses against a future exposure. Passive immunization involves
administration of preformed antibodies to an individual who is already exposed or is about to be
exposed to an antigen.
Vaccines
Active immunization, vaccination, involves administration of an antigen as a whole, killed
organism, attenuated (live) organism, or a specific protein or peptide constituent of an organism.
Booster doses often are required, especially when killed (inactivated) organisms are used as the
immunogen. In the United States, vaccination has sharply curtailed or practically eliminated a
variety of major infections, including diphtheria, measles, mumps, pertussis, rubella, tetanus, and
Haemophilus influenzae type b (Dorner and Barrett, 1999; The National Vaccine Advisory
Committee, 1999).
Although most work with vaccines has been aimed at infectious diseases, we are on the threshold of
a new generation of vaccines aimed at specific cancers or autoimmune diseases (Lee et al. , 1998;
Del Vecchio and Parmiani, 1999; Simone et al. , 1999). Such immunizations may provide complete
or limited protection from disease. Because T cells optimally are activated by peptides and
costimulatory ligands that both are present on APCs, one approach for vaccination has consisted of
immunizing patients with APCs expressing a tumor antigen. The first generation of anticancer
vaccines used whole cancer cells or tumor-cell lysates as a source of antigen in combination with
various adjuvants, relying on APCs in the host to process and present tumor-specific antigens
(Sinkovics and Horvath, 2000). These anticancer vaccines resulted in occasional clinical responses

and are being tested in prospective clinical trials. The second generation of anticancer vaccines
utilized specific APCs incubated ex vivo with antigen or transduced to express antigen and
subsequently reinfused into patients. Preclinical studies have shown that, when laboratory animals
are immunized with dendritic cells previously pulsed with MHC class I–restricted peptides derived
from tumor-specific antigens, pronounced antitumor cytotoxic T-lymphocyte responses and
protective tumor immunity can be generated (Tarte and Klein, 1999). Finally, multiple studies have
revealed the efficacy of DNA vaccines in small and large animal models of infectious diseases and
cancer (Lewis and Babiuk, 1999; Liljeqvist and Stahl, 1999). The advantage of DNA vaccination
over peptide immunization is that it permits generation of entire proteins enabling determinant
selection to occur in the host without having to restrict immunization to patients bearing specific
HLA alleles. However, a safety concern about this technique is the potential for integration of the
plasmid DNA into the host genome with the possibility of disrupting important genes and thereby
leading to phenotypic mutations or carcinogenicity. A final approach to generate or enhance
immune responses against specific antigens consists of infecting cells with recombinant viruses that
encode the protein antigen of interest. Different types of viral vectors that can infect mammalian
cells, such as vaccinia, avipox, lentivirus or adenovirus, have been used.
Immune Globulin
Passive immunization is indicated when an individual is deficient in antibodies because of a
congenital or acquired immunodeficiency, when an individual with a high degree of risk is exposed
to an agent and there is inadequate time for active immunization (measles, rabies, hepatitis B), or
when a disease is already present but can be ameliorated by passive antibodies (botulism,
diphtheria, tetanus). Passive immunization may be provided by several different products (Table
53–2). Nonspecific immunoglobulins or highly specific immunoglobulins may be provided based
upon the indication. The protection provided usually lasts from 1 to 3 months. Immune globulin is
derived from pooled plasma of adults by an alcohol-fractionation procedure. It contains largely IgG
(95%) and is indicated for antibody-deficiency disorders, exposure to infections such as hepatitis A
and measles, and specific immunologic diseases such as immune thrombocytopenic purpura and
Guillain-Barré syndrome (Ballow, 1997; Jordan et al. , 1998a; Jordan et al. , 1998b). In contrast,
specific immune globulins ("hyperimmune") differ from other immune globulin preparations in that
donors are selected for high titers of the desired antibodies. Specific immune globulin preparations

are available for hepatitis B, rabies, tetanus, varicella-zoster, cytomegalovirus, and respiratory
syncytial virus. Rho(D) immune globulin is a specific hyperimmune globulin for prophylaxis
against hemolytic disease of the newborn due to Rh incompatibility between mother and fetus. All
such plasma-derived products carry the theoretical risk of transmission of infectious disease.
Rho(D) Immune Globulin
The commercial forms of Rho(D) immune globulin (Table 53–2) are IgG containing a high titer of
antibodies against the Rh(D) antigen on the surface of red blood cells. All donors are carefully
screened to reduce the risk of transmitting infectious diseases. Fractionation of the plasma is
performed by precipitation with cold alcohol followed by passage through a viral clearance system
(Bowman, 1998; Contreras, 1998; Lee, 1998).
Mechanism of Action
Rho(D) immune globulin acts by binding Rho antigens, thereby preventing sensitization (Peterec,
1995). Rh-negative women may be sensitized to the "foreign" Rh antigen on red blood cells via the
fetus at the time of birth, miscarriage, ectopic pregnancy, or any transplacental hemorrhage. If the
women go on to have a primary immune response, they will make antibodies to Rh antigen that can
cross the placenta and damage subsequent fetuses by lysing red blood cells. This syndrome, called
hemolytic disease of the newborn, is life threatening. The form due to Rh incompatibility is largely
preventable by Rho(D) immune globulin.
Therapeutic Use
Rho(D) immune globulin is indicated whenever fetal red blood cells are known or suspected to have
entered the circulation of an Rh-negative mother unless the fetus is known also to be Rh-negative.
The drug is given intramuscularly. The half-life of circulating immunoglobulin is approximately 21
to 29 days.
Toxicity
Discomfort at the site of injection and low-grade fever have been reported. Systemic reactions are
extremely rare, but myalgia, lethargy, and anaphylactic shock have been reported. As with all
plasma-derived products, there is a theoretical risk of transmission of infectious diseases.
Prospectus
Throughout the 1990s, most transplant centers employed some combination of immunosuppressive