Immunomodulatory Agents

IMMUNOMODULATORY AGENTS Fred Farris, PhD I. THE IMMUNE RESPONSE Overview • Evolved to discriminate “self” from “non-self” • Two subsystems comprise Immune System Innate Immunity: • Natural immunity • Primitive • Needs no priming • Low affinity • Broadly reactive • Acts quickly and wanes Adaptive Immunity: • Learned immunity • Antigen specific • Primed by antigens • High affinity • Specific • Develop slowly with memory Major Effectors of Immunity Innate Immune System Adaptive Immune response • Complement • B Lymphocytes • Granulocytes • T Lymphocytes • Monocytes/macrophages • Natural Killer Cells • Basophils Cells of the Immune System Lymphoid Cells • Originate during hematopoiesis from Common Lymphoid Progenitor in bone marrow • B Cells mature in Bone Marrow • T cells mature in thymus • • • • • • Bone marrow and thymus are primary lymphoid organs Following maturation, they migrate to secondary lymphoid tissues Average human has 1012 lymphoid cells Lymphoid tissue represents 2% of total body weight Lymphoid cells represent 20% of body’s while cells Lymphocytes express specific cell surface makers (Clusters of Differentiation – CDs) on their cell surface that can be used to distinguish various cell subsets. B Lymphocytes Make Antibodies • B refers to “bursa of Fabricius” an organ where B cells mature in birds • Unique in their ability to secrete immunoglobulins • Development occurs in various stages, ending in Plasma Cells “immunoglobulin factories" • Activated B cells display immunoglobulins M and D on their surface but secrete primarily IgM as antibodies • A single B cell has 1.5x1015 antibodies on its surface Antibodies Antibodies • Y-shaped structure consisting of two identical light and heavy chains – connected by disulfide bonds • Both light (L) and heavy (H) chains have constant and variable regions • The Fab region of the complex varies according to the binding specificity of the antibody Classes of Immunoglobulins ◦ Five different classes of immunoglobulins defined by five different types heavy chain proteins in the Fc region ◦ The constant region confers on each class of antibody its effector function ◦ Each heavy chain protein differs in its antigenic reactivity, carbohydrate content and biological function Classes of Immunoglobulins Molecules that Recognize Antigens • Acquired Immune response depends on ability of two types of molecules to recognize foreign antigens • Immunoglobulins of B cells • T Cell antigen receptors (TCRs) • Antigen receptors of both T and B lymphocytes belong to the immunoglobulin supergene family and are derived from a common ancestor • Immunoglobulins are composed of two identical heavy (H) chains and two identical light (L) chains • TCR has an antigen binding portion consisting of either α and β chains or γ and δ, and ε chains T Lymphocytes Act As: T Cell Recognition of Antigens Cytokines Four particularly classes of cytokines: 1. Interleukins 2. Interferons • Type 1 • Type 2 3. Colony Stimulating Factors 4. Tumor Necrosis Factor Interleukins • A class of cytokines secreted by lymphocytes (and other cells) that promote development and maturation of T- and B-cell populations • Typically characterized by compact globular form dominated by a four α-helix bundle arranged anti-parallel • Vary in MW from 12,000 to 18,000 • Human genome encodes for more than 50 interleukins and related proteins Interleukins Interferons • First cytokines discovered • Glycoproteins produced by lymphocytes (and other cells) • Involved in actions against infections • Can act directly on infected cells to inhibit viral replication • More than 20 interferons and related molecules known • Typically contain 5 α-helices (anti-parallel) with a MW of 12.000 to 160,000 Interferons Colony Stimulating Factors • Glycoproteins that support hematopoietic colony formation. • They influence survival, proliferation and maturation of hematopoietic progenitor cells and regulate activities of the mature effector cells. • There are three lineage-specific CSFs, • Granulocyte colony-stimulating factor (G-CSF), • Monocyte– macrophage colony-stimulating factor (M-CSF) • Erythropoietin • There are two multipotential CSFs: • IL-3 • GM-CSF. Tumor Necrosis Factor (TNF) • Two isoforms - TNF-α and TNF-β of similar size and structure (185amino-acid glycoproteins) and act on same receptors • Proinflammatory cytokines first isolated in 1975 • TNF-α is expressed ubiquitously, TNF-β expressed only in immune cells • Causes necrosis of some tumors but stimulates growth of others. • Major sources are endotoxin-activated mononuclear phagocyte, endothelium after tissue damage, antigen-stimulated T cells, activated NK cells and activated mast cells • Increased concentrations of TNF cause inflammation, tissue destruction, expression of adhesion molecules, modulation of tumor growth, apoptosis II. IMMUNOSUPPRESSION III. IMMUNOTHERAPY AND THE NATURE OF COSTIMULATION AND INHIBITION Regulation of T-Cell Responses • Multiple costimulatory and inhibitory molecules interact to regulate T-cell responses • Immune activation requires two signals that emanate from the interaction of membrane proteins on APCs and T Cells • Two signals are required for T-Cell activation • Presentation of an antigen ligand to the TCR and a signaling by an additional ”costimulatory” pair A B C • Cyclosporine and tacrolimus bind to Immunophilins (FKBP) • Drug-FKBP complex inhibits calcineurin and inhibits uptake of NFAT into nucleus • NFAT = Nuclear factor of Activated T-Cells • NFAT involved in IL2 transcription in Tcells and muscle and NS development • Sacrolimus (Rapamycin) works downstream from FKBP • Drug-FKBP complex inhibits mTOR • mTOR = Mammalian Target of Rapamycin • mTOR regulates cell proliferation and apoptosis • MMF and azathioprine inhibit nucleic acid synthesis • Inhibit T-cell proliferation • Antibody Muroomunab (OKT3) inhibits TCR function via interaction with CD3 IV. IMMUNOSUPPRESSANTS AND BIOLOGICS A. Glucocorticoids Glucocorticoids: Overview • Twenty-one carbon steroids synthesized by the adrenal cortex • Possess multiple biological functions affecting numerous tissues and organ systems, including immunosuppressive effects • Used as immunosuppressants since the 1960s, cortisone being the first of the steroid immunosuppressant identified • Used in combination with other immunosuppressive agents • Due to serious effects, they are typically used short term, switching to steroid-free immunosuppressive regimens Glucocorticoids: Structures Prednisone Methylprednisolone Prednisolone Dexamethasone Glucocorticoids – Mech. of Action • Glucocorticoids inhibit all phases of inflammation by induction of lipocortin-1 (annexin A1) synthesis. • Lipocortin-1 is an anti-inflammatory protein that: • Binds to cell membranes, preventing phospholipase A2 access to arachidonic acid, resulting in diminished eicosanoid production. • Inhibits cyclooxygenase activity • Binds to leukocyte membrane receptors, affecting adhesion, chemotaxis, respiratory burst and phagocytosis. • Inhibits release of inflammatory mediators from mononuclear and polymorphonuclear phagocytes Glucocorticoids - Mech. of Action (2) • Glucocorticoids suppress acquired or cell-mediated immunity via inhibition of various cytokines, including: • IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8 and IFN-γ. • IL-2 inhibition is the most crucial effect in immunosuppression, and results in inhibition of T-cell proliferation and activation of cytolytic T cells • Glucocorticoids also suppress humoral immunity by inhibiting B-cell clonal expansion and antibody synthesis - again mediated via inhibition of expression IL-2 and IL-2 receptors by B cells • Glucocorticoid–receptor complex inhibits NF-κB which results in increased apoptosis of activated cells. Glucocorticoids: ADME ◦ Administered orally, intravenously or intramuscularly. ◦ Absorbed locally at the site of administration including skin, eye and respiratory tract ◦ Prolonged local exposure could eventually produce systemic effects. ◦ Most corticosteroids are bound to two plasma proteins, transcortin (corticosteroid-binding globulin, CBG) and albumin ◦ At higher doses, when all the plasma-binding protein sites are saturated, corticosteroids could exist in unbound form. ◦ Hepatic metabolism results in primarily inactive compounds but glucuronide or sulfate formation can occur. These are primarily excreted in urine Glucocorticoids: Toxicity • Extensive glucocorticoid use often results in disabling and life-threatening adverse effects, including: • • • • • • Growth retardation in children Avascular necrosis of bone Osteopenia Increased risk of infection Poor wound healing Hypertension B. Calcineurin Inhibitors Calcineurin • A calcium and calmodulin dependent serine/threonine protein phsophatase • Activates Nuclear Factor of Activated T-Cell Cytoplasm (NFATc), via dephosphorylation; which in turn upregulates expression of IL-2 Calc. Inhibitors: Overview • The most effective immunosuppressive drugs in routine use are the calcineurin inhibitors, cyclosporine and tacrolimus • Cyclosporine (A) is a cyclic 11 amino acid polypeptide produced by the fungus Beauveria nivea • Tacrolimus is a macrolide antibiotic produced by Streptomyces tsukubaensis • Cyclosporine and tacrolimus bind to the immunophilins, cyclophilin and FKBP-12, respectively; resulting in interaction with calcineurin and blockage of its phosphatase activity Cyclosporine • Discovered by J.F. Borel in 1976 as a result of a Swiss pharmaceutical companies’ program to discover new antibiotics from fungal metabolites • Originally isolated from the fungus Tolypocladium inflatum Gams, and thought to have antifungal activity, but its Immunoregulatory activities were discovered rapidly • Cyclosporine is hydrophobic; insoluble in water but soluble in organic solvents T-Cell Activation Cyclosporine: MOA • Cyclosporine enters cell and forms complex with cyclophilin • Complex binds to calcineurin and inhibits dephosphorylation of NFATc • The phosphorylated form of NFATc cannot be transported into the nuclus • The NFATc-NFATn transcription factor fails to form, inhibiting IL-2 synthesis • Inhibits T-Cell activation Tacrolimus: MOA • A 23-membered lactone chain isolated in 1984 from Streptomyces tsukubaensis, • Although it was originally found in a soil fungus, it is a macrolide antibiotic, and its name is derived from “Tsukuba macrolide immunosuppressant.” Tacrolimus: MOA • Tacrolimus suppresses peptidylprolyl isomerase activity by binding to the immunophilin FK506-binding protein-12 (FKBP12) • The tacrolimus–FKBP-12 complex binds to calcineurin and inhibits calcineurin phosphatase activity. • Ultimate action similar to cyclosporine Calcineurin Inhibitors: Mechanism • Inhibit T-cell activation by inhibiting calcineurin • Bind to immunophilins; Tacrolimus (FKBP-12) and Cyclosporine (Cyclophilin) • The Drug-Immunophilin complex binds to calcineurin,, inhibiting Ca++-stimulated dephosphorylation of the cytosolic component of NFAT • Dephosphorylated, cytoplasmic NFAT translocates to nucleus acts to stimulate production of IL-2 • When phosphorylated – IL-2 production is not stimulated • IL-2 is essential for proliferation of T-Cells Calcineurin Inhibitors: ADME • Highly lipophilic and distribute to adipose deposits of the body • Absorption from GIT is incomplete and variable (10% to 90%) • Tacrolimus volume of distribution based on blood is 1.0 to 1.5 L/kg; based on plasma it is 30 L/kg • Cyclosporin volume of distribution based on plasma is 3.0 – 5.0 L/k • Hepatic metabolism is extensive (especially via CYP3A) with some active metabolites • Excretion is primarily via bile and feces with less than 10% urinary excretion of parent drug • Tacrolimus is reported to decay with a half-time of 12 hr • Cyclosporine decays via biphasic profile; terminal half-time of 5 – 18 hr Calcineurin Inhibitors: Toxicity • Nephrotoxicity, neurotoxicity, hypertension, hyperglycemia and diabetes have been reported • As with other immunosuppressive agents, there is increased risk of secondary tumors and opportunistic infection Calcineurn Inhibitors: Interactions • Because of potential nephrotoxicity, blood levels and renal function should be monitored closely • Coadministration of cyclosporine and tacrolimus results in additive or synergistic nephrotoxicity; delays of at least 24 hr is required when switching a patient between these drugs • Both cyclosporine and tacrolimus are metabolized via CYP3A; monitor use of other drugs metabolized by these enzymes when used simultaneously • Grapefruit juice inhibits CYP3A and can increase blood levels of tacrolimus and/or cyclosporine C. Antiproliferative and Antimetabolic Drugs Antiproliferative and Antimetabolic Drugs 1. Sir0limus (RAPAMUNE) 2. Everolimus (AFINITOR) 3. Azathioprine (IMURAN) 4. Mycophenolate Mofetil (CELLCEPT) 5. Fingolimod (GILENYA) 6. Terifluomide (AUBAGIO) 1. Sirolimus: Overview Sirolimus or rapamycin (RAPAMUNE) is a lipophilic macrocylic lactone from the fungus Streptomyces hygroscopicus 1. Sirolimus: Mechanism of Action • Inhibit T-lymphocyte activation and proliferation downstream of the IL-2 and other T-Cell growth factor receptors • Like tacrolimus and cyclosporine, sacrolimus complexes with an immunophilin – FKBP-12 • This complex inhibits the kinase mTOR, which is a key enzyme in cell progression at the G1 → S phase of the cell cycle 1. Sirolimus: Mechanism of Action 1. Sirolimus: Pharmacokinetics • Orally administered is absorbed rapidly and plasma levels peak at about 1 hr after single oral dose • Systemic availability is about 15% but kinetics are linear • Extensively metabolized by CYP3A4 • Bulk of excretion is via feces • Some metabolites have minor activity but parent molecule accounts for 90% of immunosuppressive effect • Blood half-time after multiple doses in stable renal transplant patients is 62 hr 2. Everolimus: Structure [40-O-(2-hydroxyethyl)-rapamycin] 2. Everolimus: Overview • [40-O-(2-hydroxyethyl)-rapamycin] • FDA approved treatment of astrocytoma, breast cancer, kidney and liver transplant rejection prophylaxis, pancreatic neuroendocrine tumor, renal angiomyplipoma, and renal cell cancer • Closely related to sirolimus but shorter half-time – thus, shorter time to reach steady-state • Toxicities and interactions similar to sirolimus 3. Azathioprine: Structure • An imidazolyl (univalent imidazole radical) derivative of 6-mercaptopurine • A purine antimetabolite Imidazole 3. Azathioprine: Mechanism of Action • Following exposure to nucleophiles such as glutathione. Azathioprine is cleaved to 6mercaptopurine • 6-mercaptopurine is converted to additional metabolites that inhibit de novo purine synthesis • Appears to be a more potent immunosuppressant than 6-mercaptopurine 3. Azathioprine: Pharmacokinetics • Well absorbed following po dosing • Peak plasma levels at 1-2 hr following single dose • Half-time for azathioprine is 10 min; Half-time for 5mercaptopurine is 1 hr; Other metabolites have halftimes of up to 5 hr • Blood level are of limited predictive value because of multiple metabolites 3. Azathioprine: Toxicity/Interactions • Major toxicity Is bone marrow suppression • Other side effects include, increased susceptibility to infections, increased risk of neoplasia, hepatic and GI toxicity • Allopurinol blocks activity of Xanthine oxidase, an important enzyme in catabolism of azathioprine metabolites. Thus, avoid coadministration of azathioprine and allopurinol • Coadministration of azathioprine and other myelosuppressive agents should be monitored 4. Mycophenolate Mofetil: Structure • Mycophenolate mofetil is the 2-morpholinoethyl ester of Mycophenolic Acid (MPA) 4. Mycophenolate Mofetil: Mechanism of Action • Mycophenolate mofetil is a prodrug that is rapidly hydrolyzed to the active drug MPA • MPA is a selective, noncompetitive, reversible, inhibitor of inosine monophosphate dehydrogenase (IMPDH), an enzyme in the de novo synthetic pathway for guanine nucleotides • B and T lymphocytes are highly dependent on this pathway for cell proliferation • MPA thus selectively inhibits lymphocyte proliferation and functions, including antibody formation, cellular adhesion, and migration 4. Mycophenolate Mofetil: Kinetics • MMF undergoes rapid and complete hydrolysis to MPA after oral or IV administration • MPA is then metabolized to the inactive glucuronide MPAG • Half-time of MMF is a few minutes\Half-time of MPA is 16 hr • Most (87%) MMF is excreted in the urine as MPAG • Plasma levels of MPA and MPAG are increased in patients with renal insufficiency 4. Mycophenolate Mofetil: Toxicity • The principal toxicities of MMF are GI and hematologic; leukopenia, pure red cell aplasia, diarrhea, and vomiting • Tacrolimus in combination of MMF has been associated with activation of polyoma viruses such as BK viruses, which can cause interstitial nephritis • The use of mycophenolate in pregnancy is associated with congenital anomalies and increased risk of pregnancy loss 4. Mycophenolate Mofetil: Interactions • Tacrolimus delays elimination of MMF by impairing the conversion of MPA to MPAG and may enhance GI Toxicity • Coadministration with antacids containing aluminum and magnesium hydroxide should be avoided because they decrease absorption of MMF • MMF should not be administered with cholestyramine or other drugs that affect enterohepatic circulation. Such agents decrease plasma MPA levels, probably by binding free MPA in the intestines • Acyclovir and ganciclovir may compete with MPAG for tubular secretion 5. Fingolimod (GILENYA): Overview • First agent in the new class of small molecules, the S1P-R agonists • It is an , S1P-R prodrug that reduces recirculation of lymphocytes from the lymphatic system to the blood and peripheral tissues, thereby shunting lymphocyte away from inflammatory lesions and organ grafts 5. Fingolimod: Mechanism of Action • Specifically and reversibly causes sequestration of host lymphocytes into the lymph nodes and Peyer patches and away from the circulation – protecting lesions and grafts from T-Cell mediated attack • Does not impair T-Cell or B-Cell function • Sphingosine kinase 2 phosphorylates fingolimod and the phosphorylated product is a potent agonist at S1PRs – producing altered lymphocyte traffic 5. Fingolimod: Toxicity • Lymphopenia, is the most predictable and common side effect; this effect reverses on discontinuation of dosing • A negative chronotropic effect on the heart has been observed, with the first does affecting 30% of patients; heartrate returns to normal within 48 hr, and remains at baseline 6. Teriflunomide • Blocks de novo synthesis of pyrimidines by specific noncompetitive, reversible inhibition of the mitochondrial enzyme dihydro-orotate dehydrogenase (DHOD) • DHOD is highly expressed in proliferating lymphocytes • Teriflunomide mediated blockade of pyrimidine synthesis interrupts the cell cycle in S phase and exerts a cytostatic effect on proliferating T and b cells – this limits their involvement in the inflammatory process (used with MS) D. Biologicals: Antibodies, Cytokines, and Fusion Proteins 1. Antithymocyte Globulin (ATG) • A purified gamma globulin from the serum of rabbits immunized with human thymocytes • One of many immune globulin preparations used therapeutically, generally for passive immunization • Contains cytotoxic antibodies that bind to CD2, CD3, CD4, CD8, CD11a, CD18, CD44, CD45, and HLA class I and II molecules on the surface of human T-lymphocytes • These antibodies deplete circulating lymphocyte by direct cytotoxicity and block lymphocyte function • Polyclomal antibodies are xenogenic proteins that can elicit major side effects, including fever and chills ith the potential for hypotension 2. Anti-CD3 Monoclonal Antibodies a. Muromonab-CD3 • CD3 is a component of the TCR complex on the surface of human T-lymphocytes • This drug is an anti CD-3 antibody that targets the CD3 chain of the TCR, inducing endocytosis and T-cell inactivation and removal through phagocytosis 2. Anti-CD3 Monoclonal Antibodies a. Muromonab-CD3 (ORTHOCLONE) • CD3 is a component of the TCR complex on the surface of human T-lymphocytes • This drug is an anti CD-3 antibody that targets the CD3 chain of the TCR, inducing endocytosis and T-cell inactivation and removal through phagocytosis 2. Anti-CD3 Monoclonal Antibodies b. Humanized Anti-CD3 • Anti-CD-3 mAbs humanized to minimize the occurannce of antiantibody response and mutated to prevent binding to Fc receptors 3. Anti-CD25 Monoclonal Antibodies a. Daclizumab (ZINBRYTA) and Basiliximab (SIMULECT): Overview • Daclizumab is a humanized murine IgG1 chimeric mAb. Basiliiximab is a murine-human cchimeric mAb • Both are licensed for use in conjugation with cyclosporine and corticosteroids for the prophylaxis of acute organ rejection in patients receiving renal transplants 3. Anti-CD25 Monoclonal Antibodies a. Daclizumab and Basiliximab: Mechanism • Anti-CD25 mAbs bind with high affinity to the αsubunit of IL-2 receptor, acting as antagonists by inhibiting T-cell activation and proliferation • Daclizumab has a lower affinity but longer half-time (20 days) than does Basiliximab (7 days) • The long half-time for Daclizumab results in saturation of the IL-2Rα on circulating lymphocytes for up to 120 days after transplantation 3. Anti-CD25 Monoclonal Antibodies a. Daclizumab and Basiliximab: Toxicity • Basiliximab and Daclizumab seem relatively safe, with most clinical trials reporting adverse reactions comparable to placebo • Anaphylactic reactions and opportunistic infections may occur • Drug interactions appear to be minor 4. Anti-CD52 Monoclonal Antibodies a. Alemtuzumab: Overview • Alemtuzumab binds to the CD52 protein that is widely expressed on B-cells and T-cells, as well as macrophages, NK cells, and granulocytes • Binding induces an antibody dependent lysis of the cells, and an associated profound leukopenia that may last for more than a year 4. Anti-CD52 Monoclonal Antibodies a. Alemtuzumab: Toxicity • Most common adverse effect is neutropenia • About half of patients also experience thrombocytopenia and anemia • Another effect is autoimmune hemolytic anemia 5. Anti-CD20 Monoclonal Antibodies a. Belimumab (BENLYSTA): A mAb that targets BLYS b. Rituximab (RITUXAN): Depletes circulating mature Bcells c. Ocrelizumab (OCREVUS): Depletes circulating mature B-cells 6. Anti-CD152 Agents a. Belatacept (NULOJIX): Overview • A fusion protein composed of a modified Fc fragment of a human immunoglobulin linked to the extracellular domain of the CTLA4 (CD152) of a Tlymphocyte • Second generation CTLA4-Ig has two amino acid substitutions that increase affinity for CD80 (2-fold_ and CD86 (4-fold), yielding a 10-fold increased potency 6. Anti-CD152 Agents a. Belatacept (NULOJIX): Mechanism • Belatacept is a selective T-lymphocyte costimulation blocker that binds to cell surface costimulatory ligands CD80 and CD86 on APCs, interrupting ther interaction with CD28 Tlymphocytes • Inhibition of signal, inhibits T-cell activation, promoting anergy and apoptosis 6. Anti-CD152 Agents a. Belatacept: Kinetics & Toxicity • Kinetics are linear for normal therapeutic dosing with half-time of 11 days • Generally well tolerated 7. Anti-TNF Agents Overview • Tumor Necrosis Facor Alpha is a proinflammatory cytokine that has been implicated in the pathogenesis of several immune-mediate intestinal, skin, and joint disease • Diseases associated with elevated TNF-α include, rheumatoid arthritis and Crohn’s disease 7. Anti-TNF Agents a. Infliximab (RIMICADE)