Antibodies and Biological Products

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Chapter 6 Antibodies and Biological Products

Abbreviations
APC	Antigen presenting cell
CNS	Central nervous system
CSF	Colony-stimulating factor
CTL	Cytolytic T lymphocyte
G-CSF	Granulocyte colony-stimulating factor
GM-CSF	Granulocyte-macrophage colony-stimulating factor
IFN	Interferon
Ig	Immunoglobulin
IL	Interleukin
IM	Intramuscular
IV	Intravenous
M-CSF	Macrophage colony-stimulating factor
MHC	Major histocompatibility complex
MS	Multiple sclerosis
NFAT	Nuclear factor for activated T cells
SC	Subcutaneous
Th	T-helper (cells)
TNF	Tumor necrosis factor

Therapeutic Overview

The immune system protects the host from invading organisms and growing neoplastic cells through interaction of a wide variety of cell types and secreted factors while sparing host cells. Alterations to this highly regulated system can tip the delicate balance of host defense toward immune reactions against “self” proteins and generation of autoimmune diseases. Robust immune reactions against foreign antigens may also lead to hypersensitivity (allergic) reactions. There are now many agents with different mechanisms of actions, targets, and side-effect profiles that can be used for treatment. These drugs are in two general categories:

• Immunosuppressive/anti-inflammatory agents

• Immunostimulatory agents

The goal in the development of these agents has been to increase specificity for the immune system and minimize toxicity toward other organs. Another goal has been to minimize nonspecific immune suppression or enhance select components to obtain the desired effect while avoiding decreases in host resistance or autoimmune diseases.

Most drugs to date have targeted suppression of the immune response. The antiproliferative/antimetabolic agents such as azathioprine, cyclophosphamide, and methotrexate were developed as anticancer drugs, while the glucocorticoids, which suppress the immune response and inhibit inflammatory processes, were developed for hormonal disorders. However, neither of these drug classes is particularly selective for the immune system, and they all produce significant toxicity.

Increased selectivity for the immune system was achieved with development of the calcineurin inhibitors cyclosporine, tacrolimus, and rapamycin, and with mycophenolate mofetil, which has greater selectivity on lymphocyte proliferation than other antiproliferative immunosuppressive agents. More recently, biological

compounds such as monoclonal antibodies against cytokines, receptors, and specific immune cell antigens, as well as recombinant cytokines and cytokine receptor antagonists have provided greater selectivity and less toxicity. These drugs have made important contributions to the treatment of autoimmune diseases such multiple sclerosis (MS) and rheumatoid arthritis.

Although the immune system has redundant processes for host resistance, current immunosuppressive/anti-inflammatory therapy is still limited by an increased risk of opportunistic infections and tumors. The goal is to suppress the immune response against a specific antigen without compromising the response to other antigens (e.g., bacterial, viral proteins). Glatiramer acetate uses this approach to down regulate the specific immune response in the MS disease process. Many other drugs with an antigen-specific target are being explored and developed.

Immunostimulatory drugs currently approved are primarily human recombinant cytokines for the treatment of viral infections and cancer. Cytokines such as interferon-γ (IFN-γ), IFN-α, and interleukin-2 (IL-2) stimulate the immune system to kill bacteria, virally infected cells, and tumor cells. The cytokines granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-11 enhance the growth of hematopoietic cells from bone marrow.

The benefits of these agents are shown in the Therapeutic Overview Box.

Therapeutic Overview
Immunosuppressive/anti-inflammatory drugs
Prevent or modulate immune-mediated organ/tissue transplantation rejection
Inhibit initiation and/or progression of autoimmune diseases
Immunostimulatory drugs
Enhance immune responses against infectious disease (viral, bacterial, fungal)
Enhance immune responses against neoplastic cells
Stimulate development of immunocompetent cells from bone marrow

Mechanisms of Action

Immune Response

The role of the immune system is to recognize and remove invading microorganisms and tumor cells, while ignoring host cells, through innate and acquired immune responses. When exposed to an invading organism, nonspecific innate immunity, comprising the first line of defense, is immediately called into action. The antigen-specific responses of acquired immunity also develop in tandem to more effectively remove the organism. When re-exposed to the invading organism, antigen-specific cells of the acquired immune response are called back into action for a more rapid and effective response. This memory and the ability to specifically recognize antigens differentiates acquired from innate immunity. Key differences between innate and acquired immune responses are listed in Table 6-1.

TABLE 6–1 Characteristics of Innate and Acquired Immune Responses

Characteristic	Innate Immunity	Acquired Immunity
Onset of action	Immediate	Days to weeks
Mechanism of antigen recognition	Receptors that recognize common molecules on microbes and viruses	Unique antigen specific receptors (T-cell receptor, B-cell receptor)
Cell types/factors	Macrophages, neutrophils, mast cells, natural killer cells, complement, interferon	Antigen presenting cells, T lymphocytes, B lymphocytes

Innate Immunity

With bacterial infections, complement and phagocytic cells (macrophages and neutrophils) play a vital role in producing a local inflammatory response that leads to killing and removal of the invading microbes. Complement can be activated by antigen-antibody complexes or by natural substances such as bacterial cell wall components to produce split products that activate and recruit macrophages and neutrophils. The two pathways converge on a single final pathway in which complement components assemble into a pore-forming membrane attack complex that can directly lyse invading cells. Macrophages are activated and phagocytize the microbes, producing a variety of cytokines and chemoattractants (chemokines). The cytokines activate neighboring cells and recruit neutrophils that kill and remove the invading microbe. For viral infections, IFN is produced immediately by fibroblasts and macrophages. IFNs directly inhibit viral replication and activate natural killer cells to lyse virally-infected cells.

Acquired Immunity

Acquired immunity is commonly divided into humoral and cell-mediated responses. Initiation of acquired immunity initially involves antigen-specific activation of naive T cells (CD4⁺, T-helper cells). This requires participation of antigen presenting cells (APC) (dendritic cells, macrophages, and B cells) that take up and process antigens into peptide fragments. Peptides bind to major histocompatibility complex (MHC) Class II molecules within the APC and are presented to CD4⁺ T cells that possess a T-cell receptor specific for the peptide-MHC complex. The naïve T cell requires two signals to be fully activated. The first is provided by the peptide binding to the T-cell receptor, and the second from interaction of costimulatory molecules of the APC and the T cell (Fig. 6-1). Signal One in the absence of Signal Two leads to tolerance, or functional silencing of the T cell. Fully activated T cells proliferate and differentiate into effector T-helper (Th) cells that produce cytokines, such as ILs. In general, there are two types of effector Th cells: Th1 and Th2 cells. The type of cytokines produced by each Th cell determines its function. Cytokines produced by Th1 cells (IFN-γ, IL- 2, tumor necrosis factor [TNF]-α) stimulate generation of cell-mediated immune responses (Fig. 6-2), whereas Th2 cells produce cytokines (IL-4, IL-5, IL-10, IL-13) that drive formation of an antibody response (humoral immunity).

FIGURE 6–1 Molecular interactions between antigen presenting cells (APC) and T cells. The T-cell receptor (TCR) and CD4 complex on T-helper cells act as coreceptors for the immunogenic peptide associated with the MHC class II molecule. The TCR and CD8 molecules on cytotoxic T lymphocytes act as coreceptors for the immunogenic peptide associated with the MHC Class I molecule (not shown). T cells require two signals to become activated. The first signal involves the binding of the T-cell receptor and CD4 or CD8 with antigen-MHC complex. This binding results in the transduction of a signal via the CD3 molecule. The second signal is mediated by costimulatory molecules CD28 and B7. The stimulated T cell will in turn stimulate the APC via CD40 ligand-CD40 interactions. MHC class II antigens are found on dendritic cells, macrophages, B cells, and other specialized APCs. MHC class I antigens are present on all somatic cells.

FIGURE 6–2 Generation of effector Th1 and Th2 cells. APCs take up, process, and present peptide antigen to naïve CD4⁺ T-helper cells. The interaction between APC and T-helper cells results in activation and proliferation of effector Th1 or Th2 cells. Cytokines IFN- γ and IL-12 stimulate the formation of Th1 cells, while IL-4 drives the formation of Th2 cells. Th1 cells secrete IFN-γ, TNF-α, and IL-2, which result in the activation of macrophages and CTL generation. Th2 cells secrete IL-4, IL-5, and IL-10, which drives the generation of an antibody response. Th1 and Th2 cells can also secrete the same cytokines. The distinctions between these cell types are not always clear in certain human diseases.

Cell-Mediated Immunity

Cell-mediated immune responses involve Th1-mediated activation of macrophages (type IV hypersensitivity) and generation of CD8⁺ cytotoxic T-lymphocytes (CTLs). Th1 cells secrete cytokines, which recruit and activate macrophages. Macrophages are capable of killing intracellular bacteria and produce a localized inflammatory response. This also occurs with chemicals such as urushiol from poison ivy (contact hypersensitivity).

CTLs mediate antigen-specific lysis of tumor cells, virally infected cells, and graft/transplant cells. Generation of CTLs for all three functions generally involves similar mechanisms (Fig. 6-3). Naïve, precursor CTLs (pCTLs) require activation by two signals, as described for Th cells. The first is delivered by binding of peptide antigens associated with MHC Class I molecules on APCs to the T-cell receptor on CD8⁺ pCTLs. The second is provided by receptor-ligand interaction of costimulatory molecules. Th1 cells produce cytokines that stimulate dendritic cells to up regulate a costimulatory molecule that will activate antigen-stimulated CD8⁺ cells. Activated CTLs produce IL-2, which stimulates its own proliferation and differentiation. In certain situations, APCs that contain high levels of costimulatory molecules are able to activate CD8⁺ CTLs without the help of Th1 cells. Antigen recognition and binding of activated CTLs to antigen on cells result in cell lysis.

FIGURE 6–3 Cytolytic T-lymphocyte (CTL) generation against tumor cells. CTLs may be generated with or without support from Th1 cells. Tumor antigens are taken up by APCs, processed, and presented with MHC class II molecules to CD4⁺ T-helper cells. This leads to the generation of effector Th1 cells. Activated Th1 cells stimulate the upregulation of costimulatory molecules (B7) on APC and provide the second signal to antigen-activated precursor CTLs (pCTL). Activated CTLs secrete IL-2 and stimulate their own proliferation and differentiation to effector CTLs. Tumor cells are then recognized and lysed by effector CTLs.

Humoral Immunity

Th2 cells secrete cytokines that stimulate proliferation and differentiation of B cells to antibody-secreting plasma cells or to long-lived memory cells (Fig. 6-4). Specific antibodies can remove harmful foreign antigens (e.g., bacterial toxins) by binding to and neutralizing their effects. Antigen-antibody immune complexes can activate complement to elicit a local inflammatory reaction for further antigen removal by phagocytes. Once bound to foreign protein or bacteria, the Fc region of antibodies can bind to receptors on phagocytic cells, leading to internalization of the invading pathogens.

FIGURE 6–4 Generation of a humoral immune response. APCs internalize antigen through phagocytosis or antibody-mediated endocytosis (B cells). The antigen is processed, and the resulting peptides are presented on the surface to naïve T cells in the setting of MHC class II molecules. The interaction of the specific TCR: peptide/MHC pair and the costimulatory molecules B7/CD28 is necessary for full activation. The activated T cell produces IL-2, which leads to its proliferation and upregulation of CD25, a component of the IL-2 receptor. CD40L is also up regulated. In the lymphoid organs such as the lymph nodes or the spleen, the activated T cell interacts with a B cell that is specific for the same antigen and provides help to the B cell through interaction of CD40L and CD40 and by producing cytokines such as IL-4 that lead to B cell activation and proliferation. The activated B cells can differentiate into antibody-producing plasma cells or into memory cells.

Pharmacological Immunosuppression

Pharmacological approaches to immunosuppressive therapy may involve selective eradication of immunocompetent cells, similar to the selective killing of tumor cells by antineoplastic drugs (see Chapter 54), or down regulation of the immune response without deleting the target cell. In both cases the goal is to balance the activity and selectivity of the drug to optimize clinical efficacy while preventing adverse effects. The principal drugs used currently to obtain immunosuppression include glucocorticoids, antiproliferative/antimetabolite agents, calcineurin inhibitors, and biologicals. Most of these compounds are highly effective in inhibiting the immune response. However, their usefulness is limited by their severe toxicities. Therefore the different drugs are used in combination at lower doses to obtain a synergistic effect on immune responses while minimizing adverse effects. Immunosuppressive drugs are used primarily to prevent transplant rejection and treat autoimmune diseases.

Glucocorticoids

The actions of the corticosteroids are discussed in Chapter 39. Glucocorticoids are effective in treating autoimmune diseases and preventing graft rejection because of their immunosuppressive and anti-inflammatory effects. When glucocorticoids are administered, the numbers of circulating lymphocytes, basophils, and eosinophils decrease over a 24-hour period, whereas the number of neutrophils increases. In addition, changes in glucocorticoid concentrations during the normal diurnal cycle and in stressful situations correlate with decreases in circulating lymphocytes. Lymphopenia is attributed to migration of cells into extravascular spaces, with more T cells than B cells or monocytes migrating. Most cells migrate to bone marrow. High-dose glucocorticoid therapy is also known to reduce the size of lymphoid organs.

Although glucocorticoid-induced lymphopenia is well documented, its importance in immunosuppression is unclear. It is known, however, that glucocorticoids alter the immune response by directly changing immune cell function, and the macrophage is a primary target. Exposure of macrophages to glucocorticoids results in decreased IL-1 production, decreased expression of MHC class II antigens, and decreased phagocytosis of virus-infected cells, tumor cells, bacteria, and fungi. T and B cells are also directly affected, with T-cell-mediated responses affected to a greater extent.

Antiproliferative/Antimetabolite Agents

This class of drugs acts predominantly by deleting proliferating cells. Proliferation is a key step in the immune response and therefore a primary target. Although many cytotoxic agents have been used in treating cancer, a relatively small number of drugs are used in treating immune diseases. The main categories are alkylating agents, such as cyclophosphamide, and antimetabolites, such as azathioprine and methotrexate.

The structure of cyclophosphamide, its activation to phosphoramide mustard and acrolein, and its antitumor actions are discussed in Chapter 54. The ways in which the active metabolites phosphoramide mustard and acrolein alter the immune response are unclear. The mustard is believed to alkylate DNA and mediate the antiproliferative and immunosuppressive effects. This is consistent with the hypothesis that selective cytotoxic effects on B cells are attributable to a greater proliferation rate. However, the highly reactive, sulfhydryl-binding acrolein may also play an important role in the drug’s action.

The structure of azathioprine is shown in Figure 6-5. This drug is metabolized to the antiproliferative drug 6-mercaptopurine (see Chapter 54), which is further metabolized to the active antitumor and immunosuppressive thioinosinic acid inhibiting hypoxanthine-guanine phosphoribosyltransferase, which catalyzes the conversion of purines to the corresponding phosphoribosyl-5′ phosphates and the conversion of hypoxanthine to inosinic acid. This leads to the inhibition of cellular proliferation. The immunosuppressive effects of azathioprine stem from its antiproliferative actions.

FIGURE 6–5 Structures of several immunosuppressants.

The immunosuppressive effects of mycophenolate mofetil are mediated by inhibiting T and B lymphocyte proliferation through inhibition of purine synthesis. Purine nucleotides are synthesized in most cell types by the de novo or salvage pathways. Mycophenolate mofetil selectively inhibits inosine monophosphate dehydrogenase, blocking de novo synthesis of purines. Lymphocytes, unlike other rapidly dividing cell types, depend entirely on the de novo pathway for purine synthesis, thus explaining the selectivity of this agent for lymphocytes. Mycophenolate mofetil is an antimetabolite like azathioprine and is reported to have greater selectivity for T and B lymphocytes than for neutrophils and platelets. It inhibits the generation of CTLs and antibody-producing cells by inhibiting the proliferation of T and B lymphocytes. It also affects expression of adhesion molecules on lymphocytes, thereby inhibiting their binding to vascular endothelial cells, which is necessary for migration from the circulation to tissues.

Methotrexate was originally developed as an anticancer drug (see Chapter 54) but is now being used widely at lower doses in several inflammatory diseases, including rheumatoid arthritis. The immunological and antitumor mechanisms are similar. An antimetabolite, methotrexate binds and inactivates dihydrofolate reductase, leading to inhibition of the synthesis of thymidylate, inosinic acid, and other purine metabolites. Methotrexate also stimulates the release of adenosine, which inhibits stimulated neutrophil function and has potent anti-inflammatory properties.

Calcineurin Inhibitors/Immunophilin Binding Agents

Calcineurin inhibitors down regulate immune responses by inhibiting the production of IL-2 in activated T cells. IL-2 is a key driver of many immune responses and especially important in mediating organ transplant rejection. The two calcineurin inhibitors cyclosporine and tacrolimus bind to cyclophilin and FK binding protein, respectively, and the drug-immunophilin complex binds to calcineurin. This leads to dephosphorylation of nuclear factor for activated T cells (NFAT) and prevention of its translocation to the nucleus, causing down regulation of cytokine transcription (Fig. 6-6).

FIGURE 6–6 Mechanism of action of cyclosporine and tacrolimus. Transcription of various cytokines requires the transcription factor NFAT. Removal of a phosphate group from NFAT by the Ca⁺⁺/calmodulin-dependent calcineurin phosphatase results in translocation of NFAT to the nucleus, where it binds to the NFAT binding element of the promoter region of cytokine genes (IL-2, IL- 3, IL-4, GM-CSF, TNF-α, IFN-γ). This results in activation of cytokine transcription. Cyclosporine and tacrolimus bind to cyclophilin and FKBP-12 (known as immunophilins), respectively. These complexes inhibit calcineurin activity and inhibit NFAT dephosphorylation.

Cyclosporine is a cyclic endecapeptide purified from fungi (see Fig. 6-5). It primarily affects T-cell–mediated responses, whereas most humoral immune responses not requiring T cells are spared. The effectiveness of cyclosporine stems from its selective inhibition of Th cell activation. Its major effect on Th cells is inhibition of cytokine production. Decreased IL-2 production in turn leads to a decrease in IL-2 receptors and in a lack of responsiveness of CTL precursor cells. Because there is positive feedback through IL-2 production and IL-2 receptors, the decreased IL-2 production of Th cells also leads to decreased IL-2 receptors. Cyclosporine does not, however, affect the proliferative response of activated CTLs to IL-2 or the lytic activity of CTLs. Consistent with this is the observation that cyclosporine is effective only during the very early stages of antigen activation of Th cells. There is also evidence for inhibition of macrophage antigen presentation and IL-1 production by macrophages.

The cytoplasmic receptor for cyclosporine is cyclophilin, a propyl cis-trans isomerase involved in protein folding. Although cyclosporine is known to inhibit isomerase activity, that mechanism does not appear to be important in its immunosuppressive effects. Rather, the cyclosporine-cyclophilin drug complex binds to and inhibits calcineurin and inhibits translocation of the transcription factor NFAT as described previously.

The structure of tacrolimus (formerly known as FK506) is shown in Figure 6-5. Its mechanism of action is similar to that of cyclosporine (see Fig. 6-6) in inhibiting cytokine synthesis. Tacrolimus also binds to a cytosolic receptor known as FK506-binding protein, which is also a peptidylpropyl cis-trans isomerase but is distinct from cyclophilin. Like cyclosporine, the tacrolimus-FK506 binding protein complex also binds and inhibits calcineurin. The major effects of these immunosuppressive actions are summarized in Figure 6-7.

FIGURE 6–7 Primary mechanisms of action of immunosuppressive drugs. The antibody response shown in Figure 6-4 is used as an example to demonstrate the primary targets and mechanisms of action of immunosuppressive drugs.

Rapamycin (sirolimus) is structurally similar to tacrolimus and also binds to the FK506-binding protein. However, unlike the calcineurin inhibitors, rapamycin blocks B and T cell activation at a later stage. It blocks signal transduction in T cells and inhibits cell-cycle progression from G1 to S phase. Rapamycin and cyclosporine appear to act synergistically to inhibit lymphocyte proliferation.

Biologicals

Advances in biotechnology and understanding the mechanisms underlying immunologic diseases have resulted in many novel biological agents. “Biologicals” refers to a diverse group of naturally occurring compounds that are manufactured primarily by advanced biotechnology techniques and includes recombinant vaccines, blood products, cytokines, growth factors, and monoclonal antibodies. Antibodies and cytokine antagonists designed to bind to soluble proteins and surface receptors that mediate inflammatory processes and drive the immune response have demonstrated clinical efficacy in a variety of neoplastic and immune disorders. Monoclonal antibodies exert their effects, depicted in Figure 6-8, through one or more of the following mechanisms:

• Blocking the function of the target protein

• Altering the function of the target cell

• Direct induction of cytotoxicity

• Immune-mediated removal of target cells through complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, or phagocytosis

FIGURE 6–8 Strategies to inhibit cytokine action. Cytokines exert their effects through binding to cell surface receptors. Monoclonal antibodies may be developed to bind to the cytokine (a) or to the receptor (b). A soluble receptor may be used to bind to the cytokine (c) as well. A soluble receptor antagonist (d) that binds specifically to the receptor without activating it may be used to compete against the activating cytokine.

Biologicals Targeting Lymphocytes

Anti-thymocyte globulin is purified IgG obtained from rabbits immunized with human thymocytes. Intravenous (IV) administration of these polyclonal antibodies leads to rapid and profound depletion of peripheral lymphocytes and is used to prevent graft rejection. Severe side effects limit the use of these antibodies to second-line status behind calcineurin inhibitors. In addition, because of the nature of production, there is poor standardization between batches. Monoclonal antibodies, on the other hand, are well standardized. OKT3 (muromonab) is a monoclonal antibody directed against the CD3 molecule on the membrane of T cells. CD3 is associated with the T-cell antigen receptor complex involved in signal transduction to the T cell after binding of antigen (see Fig. 6-1). Muromonab is approved for treatment of acute rejection of renal transplants, but it has severe side effects and is generally reserved for patients resistant to first-line therapies.

Biologicals Targeting Cytokines

TNFα and IL-1β are pro-inflammatory cytokines implicated in the pathogenesis of inflammatory disorders such as rheumatoid arthritis (Fig. 6-9) and Crohn’s disease (see Chapter 18). They are involved in activation and proliferation of synovial cells, inducing the production of collagenases and other cytokines that lead to continued inflammation and bone resorption. Infliximab and adalimumab are antibodies that bind to soluble TNF-α and lower its level in blood. The former is a murine/human chimeric antibody, and the latter is a recombinant humanized monoclonal antibody. Etanercept is a dimeric fusion protein combining the p75 TNF receptor with the Fc portion of human IgG1.

FIGURE 6–9 TNF-α and IL-1 play a central role in rheumatoid arthritis. Although the inciting factors are unknown, activated macrophages produce TNF-α and IL-1, which activate synovial fibroblasts in the synovium of affected joints. This in turn leads to recruitment of other inflammatory cells to the joint and to release of metalloproteinases, which results in bone and cartilage degradation characteristic of the disease. TNF-α also acts in an autocrine fashion to perpetuate the inflammatory pathway. Other cytokines such as IL-6 also play important pro-inflammatory roles.

IL-1 receptor antagonist (IL-1ra) is a naturally occurring antagonist of IL-1. IL-1ra binds to the two receptor forms of IL-1 (type I and II) and inhibits binding of both IL-1α and IL-1β without stimulating the cells. Anakinra is a recombinant IL-1ra that is used for treatment of rheumatoid arthritis.

IL-2 receptor antagonists prevent IL-2 from binding to activated T lymphocytes. Unlike muromonab, which targets both resting and activated lymphocytes, IL-2 receptor antagonists target actively dividing cells by binding to the α chain of the trimolecular IL-2 receptor (CD25), which is transiently expressed only on antigen-activated T cells. There are two currently available monoclonal antibodies directed against IL-2 receptor α, basiliximab and daclizumab. Early clinical studies demonstrate efficacy in combination with calcineurin inhibitors. Although long-term data are lacking, these antibodies appear to be well tolerated.

Cytokine Biologicals

The IFNs comprise a family of cytokines produced by many cell types that produce antiproliferative, antiviral, and immunomodulatory effects. They were originally named for their ability to interfere with viral RNA and protein synthesis. IFNs are categorized as either type I (α and β) or type II (γ). The α and β forms are similar in structure and bind to the same receptors. There is little sequence homology between the γ and the other two types. Upon viral stimulation, the α form is primarily synthesized in macrophages and the β form in macrophages and fibroblasts. Type I IFNs are known to both stimulate the immune response (see below) and mediate anti-inflammatory actions (treatment of MS). MS is mediated by myelin-reactive Th1 cells that migrate into the central nervous system (CNS) and produce an inflammatory reaction. Although the exact mechanism for the effect of IFN-β on MS is not known, data indicate that it inhibits T cell migration by suppressing synthesis of chemokines and adhesion proteins. The γ form is primarily produced in T lymphocytes after stimulation with antigen or mitogens (see below).

Biologicals Targeting Hypersensitivity Mediators

Allergen binding and cross-linking of specific-IgE bound to mast cells leads to mast cell degranulation and release of various mediators (histamine, leukotrienes) involved in asthma (see Chapter 16). A monoclonal antibody specific for IgE (omalizumab) is approved for allergic asthma. Omalizumab inhibits binding of IgE to the IgE Fc receptor on mast cells, preventing antigen-induced mediator release.

Antigen-specific Immunomodulation Glatiramer Acetate

Glatiramer acetate is a drug that was specifically designed for treatment of MS with a mechanism of action different from that of IFN-β. MS is an autoimmune disease in which an immune response against myelin proteins is believed to result in a localized inflammatory response to the cells of the CNS. Glatiramer acetate is a synthetic copolymer composed of four amino acids that range from 40 to 90 amino acids in length and has a structure similar to myelin basic protein. Its administration results in the generation of glatiramer acetate-specific Th2 cells, which are recruited into the CNS, activated by myelin proteins, and produce various cytokines (e.g., IL-10) that depress immune responses to myelin basic protein. Thus glatiramer acetate has a unique mechanism of action in that it is antigen specific.

Pharmacological Immunostimulation

Immunostimulatory drugs mediate their effects by directly activating or stimulating the growth of immunocompetent cells. Given the benefits of stimulating the immune response against tumor cells or pathogens, there have been many attempts to develop immunostimulant drugs. However, only a few are efficacious without overriding toxicities. The available compounds are primarily human recombinant cytokines (excluding vaccine adjuvants).

IL-2 is secreted by helper CD4⁺ T-lymphocytes, and its primary effect is autocrine stimulation of T-lymphocyte proliferation. This results in a greater immune response against a variety of antigens. Recombinant human IL-2 has been found to be effective in treatment of certain types of cancer. The exact mechanism is unknown.

Colony-stimulating factors (CSFs) comprise a group of cytokines named for their ability to induce formation of certain types of colonies from bone marrow cells in soft agar cultures. They affect bone marrow cells at different stages of maturity. Multi-CSF (IL-3) stimulates the primitive progenitor cells that give rise to granulocytes, megakaryocytes, mast cells, macrophages, and erythrocytes. In contrast, more-differentiated progenitor cells are stimulated by G-CSF and M-CSF to proliferate and differentiate into granulocytes and macrophages, respectively. Both of these cell lineages are stimulated by GM-CSF. As cells of certain lineages mature from progenitors to more committed states, however, they become refractory to certain CSFs and sensitive to others. With exposure to a pathogen (e.g., bacteria, virus-infected cells), T cells activate and produce IL-3 and GM-CSF, whereas activated macrophages produce M-CSF, G-CSF, and GM-CSF. Activated macrophages also produce IL-1 and TNF, which stimulate production of GM-CSF, G-CSF, and M-CSF by endothelial and mesenchymal cells. In this manner the host produces more granulocytes and macrophages to combat the invading organism (Fig. 6-10).

FIGURE 6–10 Secretion of colony-stimulating factors (CSFs) in response to immune stimulation. Bacteria or other stimuli (A) activate macrophages and T cells to produce a variety of CSFs (B), which also stimulate endothelial cells to produce CSFs (C). CSFs stimulate production of macrophages and neutrophils from bone marrow (D). These cells are then able to phagocytose the bacteria (E).

IFN-α is used for the treatment of viral infections (α2b, α2a, αcon1, αn3) and tumors (α2b, α2a). These actions are attributed to direct inhibition of viral replication in host cells and inhibition of tumor cell proliferation. Inhibition of viral replication may result from induction of an enzyme that inhibits viral replication by catalyzing the breakdown of viral RNA. Anti-tumor actions appear to result from reduced oncogene expression. Both antiviral and anti-tumor actions may also be indirectly attributed to effects on innate and acquired immune responses. IFN-α activates natural killer cells to kill viral-infected and tumor cells and stimulates upregulation of MHC class I expression. Class I MHCs present antigen to CD8⁺ cytotoxic T cells that will also kill viral-infected cells and tumor cells.

IFN-γ stimulates the immune response by induction of MHC expression on dendritic cells, macrophages, and B cells, resulting in an increased ability to present antigen. Macrophages are activated by IFN-γ to increase hydrogen peroxide production, phagocytosis, and expression of Fc receptors and thereby enhance their cytocidal action. These effects are greater than those obtainable with IFN-β or IFN-α. IFN-γ also activates natural killer cells to destroy virus-infected and neoplastic cells. Thus, in contrast to type I IFNs, IFN-γ is a pro-inflammatory cytokine that helps drive cell-mediated immune responses.

A summary of selected biologicals and their targets, actions, and indications are listed in Table 6-2.

TABLE 6–2 Selected Biologicals

Pharmacokinetics

Many immunopharmacological agents have relatively narrow therapeutic indexes and are often used in combination. Therefore the combined toxicity and the effect of drug-drug interactions must be considered in choosing a safe and effective dosing regimen. In addition, biologicals have unique pharmacokinetics that greatly affect their use. Pharmacokinetic parameters for selected drugs are shown in Table 6-3. Because of their unique properties, little information is available about many biologicals.

TABLE 6–3 Selected Pharmacokinetic Parameters

Low Molecular Weight Drugs

Glucocorticoids are discussed in Chapter 39, while cyclophosphamide and methotrexate are discussed in Chapter 54. Methotrexate is used at low oral doses to treat chronic autoimmune diseases. Azathioprine is usually given IV as a loading dose on the day of transplantation, with subsequent oral maintenance doses. It is rapidly absorbed and converted to 6-mercaptopurine, which is the active drug. Most metabolites are excreted in urine. One pathway of metabolism of 6-mercaptopurine involves oxidation by xanthine oxidase, which is inhibited by allopurinol. Therefore coadministration of these drugs requires a dose adjustment.

Mycophenolate mofetil is rapidly metabolized to the active metabolite, mycophenolic acid. The mofetil moiety dramatically increases bioavailability. Mycophenolic acid is bound appreciably to serum albumin and is primarily excreted in the kidney as the glucuronide conjugate.

Cyclosporine is poorly absorbed from the small intestine (bioavailability of ~30%) and is dependent on biliary flow for absorption. It is metabolized by cytochrome P4503A, which may cause drug interactions (see Chapter 2). A major constraint in dosing is nephrotoxicity, and as a result of high variability in absorption and metabolism, levels fluctuate. Tacrolimus is 10 to 100 times more potent than cyclosporine and does not rely on bile for absorption. It is also metabolized by cytochrome P4503A, and nephrotoxicity occurs with similar frequencies as with cyclosporine. Blood concentrations for these drugs are monitored to optimize their effects.

Biologicals

Biologicals such as monoclonal antibodies and cytokines are administered only by parenteral routes including IV, subcutaneous (SC), or intramuscular (IM) delivery. The t_1/2 of the compound will depend on its stability and clearance. The primary clearance mechanism of proteins less than 70 kilodaltons is via filtration through the kidneys. Larger proteins are cleared by proteases and liver uptake. For human monoclonal antibodies, the t_1/2 is similar to that of normal immunoglobulin (up to 3 weeks).

Because biologicals can be recognized as foreign, an antibody response can develop. This can lead to the development of anti-cytokine and anti-monoclonal antibodies that could bind the compound, neutralize its activity, and enhance its clearance by immune complex uptake by the reticuloendothelial system. Given the significance of immune responses on efficacy, pharmacokinetics, and safety, all package inserts of biologicals include a section on drug immunogenicity.

Early monoclonal antibodies were entirely rodent in origin, and immune responses in humans were very robust. The presence of human anti-mouse antibodies led to rapid clearance, resulting in decreased efficacy. Muromonab administration is associated with 80% human anti-mouse antibody formation, with a resulting decrease in exposure and efficacy. Simultaneous use of low-dose cyclosporine reduces the frequency to 15%. Chimeric antibodies are produced by combining the human heavy chain constant region sequences to the mouse variable region. Basiliximab is a chimeric antibody with 25% murine content, and daclizumab has a murine content of 10%. The “humanization” of these antibodies results in prolongation of serum t_1/2. Monoclonal antibodies against TNF-α also induce antibody formation. Infliximab, a chimeric monoclonal antibody, is administered with low-dose methotrexate to decrease antibody formation. More recently, monoclonal antibodies have been designed with almost all or entirely human sequences. However, antibodies can still develop against the variable domain of the human monoclonal antibody. For example, the humanized adalimumab is associated with immune responses.

To increase the t_1/2 of biologicals, polyethylene glycol is added. Conjugation with polyethylene glycol increases the size and modifies the overall charge to decrease glomerular filtration. In addition, it is thought to decrease immunogenicity by masking antigenic sites. IFN-α2b (peginterferon α2b) and G-CSF (pegfilgrastim) have been conjugated to enhance pharmacokinetic properties. For example, the serum t_1/2 of IFN-α2b is increased from 7 to 9 hours to 40 hours after conjugation with polyethylene glycol.

Relationship of Mechanisms of Action to Clinical Response

Immunosuppression

Graft Rejection

Genetically coded antigens are the determining factor in rejection of a graft by the host. Most human studies of transplant rejection involve renal allografts. Rejection processes can be classified according to how quickly they occur. Hyperacute rejection can occur within minutes and is mediated by cytotoxic antibodies circulating in the host because of previous exposure to graft antigens. Cytotoxic antibodies to type ABO blood group antigens may mediate rejection in a mismatch. Accelerated rejection occurs in 2 to 5 days, with the mechanism being an accelerated form of the acute process, again mediated by previous exposure to graft antigens (secondary immune response). Acute rejection occurs over 7 to 21 days and is mediated by a primary response that requires effector cells to be generated. Chronic rejection occurs after about 3 months.

The immune process of graft rejection is divided into afferent and efferent stages. In the afferent stage the response is initiated by graft cells possessing MHC class II antigens (bone marrow-derived dendritic cells, Langerhans cells, certain endothelial cells) that are incompatible with the host. These cells, termed passenger cells, drain into the host lymphatics and directly stimulate T cells without the need for host APCs. Contact between circulating T cells and special antigens may also occur in the graft. It is known that tissues containing a greater burden of passenger cells are more likely to be rejected (e.g., skin and bone marrow). In addition, removal of passenger cells before transplantation dramatically decreases rejection.

The efferent stage involves activation of macrophages and T cells by various effector mechanisms to destroy the graft: antibody, CTLs, and delayed hypersensitivity. Common to all three responses is the involvement of Th cells. Special antigens on the graft can activate Th cells and the production of cytokines, which stimulate activation, proliferation, and growth of T and B lymphocytes and macrophages. In the delayed-type hypersensitivity response, Th cell-produced cytokines activate and recruit monocytes to the graft. These activated macrophages nonspecifically destroy surrounding tissue.

Autoimmune Diseases

The immune system selectively destroys infectious microbes and tumor cells through its ability to mount a response to foreign antigens while ignoring self-antigens. It is thought that the deletion or deactivation of autoreactive lymphocytes occurs during their early development in the bone marrow or thymus, or that they are functionally silenced in the circulation by regulatory cells or other external factors. If this finely regulated system malfunctions, an immune response against one’s own tissue, or an autoimmune disease, may develop. Autoimmune diseases may be broadly categorized as either organ specific or systemic. In organ-specific diseases, immune responses are mounted against antigens specific to a certain organ, with manifestations specific to that organ. Examples of organ-specific diseases and the possible targets of the immune response include Hashimoto’s thyroiditis (thyroid antigens), myasthenia gravis (acetylcholine receptor), Graves’ disease (thyroid-stimulating hormone receptor), and insulin-dependent diabetes mellitus (pancreatic β cells). In contrast, in systemic autoimmunity, immune responses are mounted against tissue components in most cell types (e.g., DNA, cytoskeletal proteins). Systemic lupus erythematosus and rheumatoid arthritis are two examples. The association of several autoimmune disorders in the same individual or in related family members points to common pathogenic mechanisms that may underlie both types of autoimmunity.

The cause of autoimmunity remains unknown, but intrinsic signaling defects in the activated lymphocytes, abnormal presentation of self-antigens, or dysregulation of the immune response by regulatory cells may all be involved. In addition, there is a striking increased susceptibility of most autoimmune diseases in women, suggesting a role for hormonal factors. Despite the different mechanisms, common pathways and cell types involved in the immune response have led to the use of immunosuppressive drugs for treatment.

Immunosuppressive Drugs

Glucocorticoids.

Many synthetic glucocorticoid derivatives (see Chapter 39) are used as immunosuppressive agents. Glucocorticoids are often administered at high doses during acute exacerbations of disease for rapid control, followed by a slow tapering and maintenance on the lowest efficacious dose to minimize toxicity. Glucocorticoids are usually administered with other immunosuppressive agents to treat graft rejection and autoimmune diseases. Many of these combinations result in a synergistic effect. This allows doses of glucocorticoids to be decreased, decreasing the risk of toxicity. Because of their anti-inflammatory actions, glucocorticoids are effective in treatment of immunological problems exacerbated by inflammatory reactions. This is especially evident in the topical use of these agents for treatment of dermatological problems such as contact hypersensitivity to poison ivy and atopic dermatitis and in treatment of asthma (see Chapter 16).

Cyclophosphamide is often administered in cases of severe manifestations of autoimmune disease such as lupus nephritis and systemic vasculitides. However, since the discovery of cyclosporine, it is now used much less to prevent rejection of grafts. As with other immunosuppressive drugs, cyclophosphamide is often given in combination with glucocorticoids. Because life-threatening toxicities may occur with cyclophosphamide, extreme care should be taken to administer only the minimal dose necessary. Humoral immune responses are more sensitive to cyclophosphamide than are cell-mediated responses. However, at high doses, both arms of the immune response are affected.

Azathioprine is approved for prevention of acute rejection of kidney transplants. Azathioprine is ineffective alone in solid organ transplantation and is commonly used with glucocorticoids and cyclosporine in triple-combination therapy. It is also often used in treatment of moderate to severe manifestations of systemic lupus erythematosus and is indicated for rheumatoid arthritis, although not as a first-line treatment. The primary targets of azathioprine are cell-mediated immune responses. Inhibition of the in vitro immune response is maximal during initiation of the response. This time-dependent action is consistent with clinical observations that azathioprine is ineffective against ongoing graft rejection. Additional in vitro investigations have revealed that azathioprine primarily affects antigen-stimulated lymphocytes, whereas unstimulated spleen cells are unaffected. Primary immune responses are suppressed by azathioprine, whereas secondary responses are not.

Mycophenolate mofetil is indicated for prophylaxis against renal transplant rejection and is used in combination with glucocorticoids. Like azathioprine, it does not inhibit cytokine production but does inhibit lymphocyte proliferation. It is also gaining wider use in the treatment of systemic lupus erythematosus, given its favorable side effect profile relative to cyclophosphamide.

Although methotrexate is a potent immunosuppressive agent, its numerous adverse effects (see Chapter 54) have limited its widespread use in treatment of immune-associated diseases. However, low-dose, weekly administered oral methotrexate is used widely to treat rheumatoid arthritis. It is also used for psoriasis and to reduce required doses of glucocorticoids in chronic vasculitis or conditions that require prolonged periods of immunosuppression.

The objective of immunosuppressive therapy is to specifically inhibit the immune response against the graft or autoantigen. However, drugs that also affect proliferating cell populations such as cyclophosphamide, azathioprine, and methotrexate may produce life-threatening bone marrow suppression. Until the early 1980s, the use of these drugs in combination with glucocorticoids was the preferred therapy. They have now been largely supplanted by cyclosporine, which usually is effective in preventing acute graft rejection (during the first 3 weeks), generally without bone marrow toxicity. Cyclosporine is also used in patients with rheumatoid arthritis unresponsive to other therapies and in patients with certain types of lupus nephritis.

Tacrolimus is indicated for prevention of acute rejection of liver transplants. Although tacrolimus and cyclosporine have similar mechanisms of action, tacrolimus has been found effective in reversing liver transplant rejection resistant to cyclosporine. It is recommended that glucocorticoids be given with tacrolimus.

Rapamycin is indicated for prevention of acute organ rejection in combination with the calcineurin inhibitors. Its main role may be to allow for lower doses of cyclosporine or tacrolimus to be used to decrease toxicity.

Muromonab is used to reverse acute allograft rejection in patients receiving other immunosuppressive drugs (rescue therapy) and to prevent acute graft rejections (induction therapy). Because of its relatively greater toxicity, and because it has not demonstrated significant benefits relative to calcineurin inhibitors as induction therapy, it is usually reserved for patients with severe steroid-resistant rejection. Rescue therapy is followed by administration of other immunosuppressants. Its usefulness in rescue therapy stems from its ability to immediately reduce the number of circulating T lymphocytes. Because it is a murine monoclonal antibody, neutralizing antibodies usually develop in patients after 10 days. However, this rarely results in allergic or anaphylactic reactions. When it is given with prednisone and azathioprine, the development of neutralizing antibodies is reduced.

IL-2 receptor antagonists such as basiliximab and daclizumab are indicated for prevention of acute organ rejection in patients receiving renal transplants. They have demonstrated efficacy when used in combination with cyclosporine and glucocorticoids. Although long-term studies are not yet available, they should allow less-toxic doses to be used.

The relative safety, early onset of symptom relief, and efficacy in patients with rheumatoid arthritis who fail to respond to methotrexate have made anti-TNF biologicals an increasingly valuable class of agents for several autoimmune diseases. Etanercept is indicated for treatment of patients with rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. Infliximab was first approved for Crohn’s disease and is also indicated for rheumatoid arthritis. Adalimumab, the most recently approved anti-TNF monoclonal antibody, is indicated for rheumatoid arthritis.

IFN-α and IFN-β were originally proposed as therapeutics for MS, based on the belief that viral infections and low interferon production contribute to its pathogenesis. Through many years of clinical study, β-IFNs (β1a and 1b) were found to be efficacious and are marketed for treatment of relapsing forms of MS to decrease the frequency of clinical exacerbations and delay physical disability.

Omalizumab is used for treatment of patients who have asthma that is not adequately controlled by inhaled glucocorticoids and who have demonstrated sensitivity to aeroallergens. Glatiramer acetate is approved for use in the reduction of the frequency of relapses in patients with relapsing-remitting MS.

Immunostimulant Drugs

IL-2 is approved for metastatic renal carcinoma and melanoma. The mechanism of action may be related to increased killing of tumor cells by immune-mediated mechanisms.

Sargramostim, a recombinant human GM-CSF, is used to stimulate bone marrow growth in patients undergoing bone marrow transplantation. Filgrastim is a recombinant human G-CSF used in patients undergoing myelosuppressive chemotherapy. G-CSF conjugated to polyethylene glycol (pegfilgrastim) is approved for the same indications as filgrastim but has a longer t_1/2 and requires less frequent administration. Human recombinant IL-11 (oprelvekin) is also available for treatment of severe thrombocytopenia produced by myelosuppressive therapy of nonmyeloid malignancies. IL-11 works in concert with IL-4 and IL-3 to stimulate hematopoiesis. Therapy with CSFs has been found to be useful in dramatically increasing levels of neutrophils, eosinophils, and monocytes with minimal side effects. It also decreases the time it takes for engraftment to occur, the need for antibiotics, and the incidence of infections in bone marrow transplant recipients.

Recombinant human IFN-α (2a or 2b) is marketed for treatment of hepatitis C, hairy cell leukemia, and AIDS-related Kaposi’s sarcoma. IFN-α2b is also indicated for chronic hepatitis B, malignant melanoma, follicular lymphoma, and condylomata acuminata (genital warts associated with human papilloma virus). Two additional α IFNs are also marketed, IFN-αcon1 (for chronic hepatitis C) and -αn3 (for condylomata acuminata). The antitumor and antiviral effects are mediated through a direct effect on tumor and viral infected cells and through stimulation of immune responses. A polyethylene glycol conjugate of IFN-α2a (peginterferon α2a) was also developed to increase its t_1/2.