8 Treating Autoimmune Bullous Skin Disorders with Biologics

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Chapter 8 Treating Autoimmune Bullous Skin Disorders 8 with Biologics R. Eming, A. Niedermeier, M. Pfütze, A. Jacobi, M. Hertl

8.1 the specificity of the targeted antigens, several clinical- Introduction ly and immune serologically distinct bullous disorders have been defined (Fig. 8.2). 8.1.1 Autoimmune Bullous Skin Disorders 8.1.1.1 Autoimmune bullous skin disorders represent a group Pemphigus of severe, chronic skin diseases which are characterized bythepresenceofautoantibodiestargetingdistinct Desmogleins (Dsg) are transmembranous components adhesion molecules of the epidermis and dermoepider- of desmosomes, adhesion units specialized in confer- mal basement membrane zone leading to a loss of ring epidermal keratinocyte adhesion, and are linked adhesive function of the target antigen(s) (Fig. 8.1). to intercellular molecules of the desmosomal plaque Theappearanceofblistersanderosionsoftheskinand/ which in turn interact with components of the cytoskel- ormucousmembranesistheleadingclinicalsignof eton (Fig. 8.1a). Among several forms of pemphigus, autoimmunebullousskindisorders(Fig.8.2).While pemphigus vulgaris (PV) and pemphigus foliaceus histopathology reveals the location of the blister for- (PF) represent the major subtypes. IgG autoantibodies mation, the detection of tissue bound autoantibodies against Dsg3 in PV and Dsg1 in PF lead to loss of des- by immunofluorescent staining of uninvolved perile- mosomal adhesion of epidermal keratinocytes and sional skin biopsies is mandatory for diagnosing auto- intraepidermal blister formation (Fig. 8.2A–C). In PV immune bullous skin disorders. Circulating autoanti- patients suffer from flaccid blister/erosions of the bodies can be visualized by indirect immunofluores- mucous membranes, primarily the oral mucosa and the cence using tissue substrates such as monkey esopha- skin. PF is characterized by crusted painful erosions gus and sodium chloride-split human skin. Based on typically of the seborrheic areas such as scalp, face,

Fig. 8.1. Schematic overview of desmosomal and hemidesmosomal autoantigens in autoimmune bullous skin disorders. Shown are the major components of desmosomes which have been identified as autoantigens in the different clinical variants of pemphigus (a) 70 8 Treating Autoimmune Bullous Skin Disorders with Biologics

Fig. 8.1. (cont.) The integrity of epidermal cohesion is largely dependent on desmosomes, plaque-like intercellular adhesion structures that connect transmembranous adhesion molecules such as the desmogleins and desmocollins with keratins of the cytoskeleton through interaction with intracellular components of the desmosomal plaque, such as desmoplakin, plakoglobin, plakophilin, envoplakin and periplakin. In addition to desmoglein 3 and 1, which are autoantigens in pemphigus, all the other aforementioned components of desmosomes have been identified as autoantigens of different clinical variants of pemphigus (b). Components of hemidesmosomes and the basement membrane are autoantigens of the pemphigoids and epidermolysis bullosa b acquisita. Basal keratinocytes adhere to the basement membrane zone by the interaction of cytoplasmic and transmembranous components of hemidesmosomes, q such as BP180, BP230 and 4 integrin, respectively, with ligands such as laminin 5 located in the dermoepidermal basement membrane zone. The intracellular hemidesmosomal components BP230 and plectin are linked to keratins of the cytoskeleton [ q and interact with the cytoplasmic domains of BP180 and 6 4 integrin which in turn interact with laminin 5 via their ectodo- mains. Type VII collagen is the major component of anchoring fibrils which link the basement membrane to the dermis by direct interaction with laminin 5 in the lamina densa of the basement membrane (modified from Hertl et al. 2006)

Fig. 8.2. Diagnostic and clinical characteristics of autoimmune bullous skin disorders. Pemphigusischaracterized bythepresenceofIgG(and occasionally IgA) specific for desmosomal target antigens (as shown here by direct immunofluorescence, (A) resulting in a loss of intraepidermal adhesion (as shown by histopathology, (B)and blisters/erosions of the mucous membranes and skin (C). In the pemphigoids, including linear IgA bullous dermatosis, IgG (or IgA) autoantibodies bind to antigens of hemidesmosomes and the dermoepidermal junction (D), resulting in a loss of subepidermal adhesion (E)andtenseblisters (F). Epidermolysis bullosa acquisita is associated with IgG (and sometimes IgA) binding to the anchoring fibrils underneath the dermoepidermal junction (G), resulting in a subepidermal loss of adhesion (H) and tense blisters with a tendency to scarring and milia formation (I). Der- matitis herpetiformis is associated with IgA deposits in the papillary dermis (the dotted line indicates the dermoepidermal junction)reactivewithepidermaltransglutaminase(K), a subepidermal loss of adhesion (L), and herpetiform blisters or pruritic papules (M) 8.1 Introduction 71 chest and upper back whereas mucosal lesions are sting-Perry pemphigoid, mucous membrane pemphi- absent in PF. In general, the treatment of pemphigus is goid or linear IgA bullous dermatosis. Mechanobullous based on immunosuppressive treatment regiments EBApresentsasanon-inflammatorydiseasewithan consisting of high dose glucocorticoids and immuno- acral distribution and skin fragility over trauma prone suppressive “steroid-sparing” adjuvants, including aza- surfaces. The blisters and erosions heal with scarring thioprine or mycophenolate mofetil. and milia formation (Fig. 8.2G–I). Especially the mechanobullous form of EBA often reveals itself refractory to high doses of systemic glucocorticoids com- 8.1.1.2 bined with immunosuppressive adjuvants. Other treat- Bullous Pemphigoid ment options include the use of colchicines, high dose In the pemphigoids, IgG autoantibodies against com- immunoglobulins or immunoadsorption. ponents of the dermoepidermal basement membrane, such as bullous antigen 180 (BP180/BP antigen 2/type 8.1.1.4 XVII collagen), BP antigen 230 (BP230/BP antigen 1), Dermatitis Herpetiformis Duhring and laminin 5 interfere with the adhesion of basal epidermal keratinocytes to the dermoepidermal basement Dermatitis herpetiformis (DH) represents a bullous or membrane zone (Fig. 8.1b). BP180 and BP230 are pruritic autoimmune disorder with subepidermal blis- transmembranous and intracellular components, ter formation which is considered to be a specific cuta- respectively, of hemidesmosomes of basal epidermal neous manifestation of celiac disease, although most keratinocytes while laminin 5 is a ligand of BP180 DH patients do not present gastrointestinal symptoms. located in the lamina lucida of the basement membrane The autoantigen of dermatitis herpetiformis, epider- zone (Fig. 8.1b). Bullous pemphigoid (BP) is the most mal transglutaminase, is targeted by autoantibodies of common autoimmune blistering disease in adults. Pro- the IgA class. Immunofluorescent staining of unin- dromal, non-bullous periods of BP may occur in which volved perilesional skin biopsies reveals granular IgA patients demonstrate pruritic eczematiform papules or deposition in the papillary dermis (Fig. 8.2K–M). DH urticarial plaques. The bullous manifestations of BP patients show an intense pruritus with eruption of ery- presentastense,fluid-filledvesiclesandbullaeonnor- thematous papules and herpetiform vesicles distribut- mal or erythematous skin in combination with urticat- ed symmetrically on the extensor surfaces. In addition ed plaques (Fig. 8.2D–F). Involvement of the oral to a gluten-free diet, the sulphonamide diamino-diphe- mucosa is rarely observed in about 10–30% of nyl sulfone (dapsone) is most commonly used in the patients. In contrast to pemphigus, the use of potent treatment of DH. topical corticosteroids such as clobetasol propionate has been shown to be effective even in the treatment of 8.1.2 generalized BP. Nevertheless, BP patients with exten- Immune Pathogenesis of Bullous Autoimmune Disorders sive disease are usually treated with systemic prednisone at doses of 0.5–1.0 mg/kg/day, which is then pro- Pemphigus and pemphigoid are considered to be pro- gressively tapered. totypic bullous disorders based on their well-characterized immune pathogenesis. Apart from pemphigus and BP, there is only circumstantial evidence that auto- 8.1.1.3 reactive T cells are present and involved in the patho- Epidermolysis Bullosa Acquisita genesis of the autoimmune bullous disorders epider- Epidermolysis bullosa acquisita (EBA) is a rare chronic molysis bullosa acquisita and dermatitis herpetiformis. subepidermal bullous disease of the skin and mucous In PV and BP, autoreactive CD4+ Tlymphocytesthat membranes characterized by the presence of autoanti- are presumably crucial in initiating the autoimmune bodies against type VII collagen, a major component of response recognize distinct epitopes of the extracellu- anchoring fibrils. There is a great diversity in the clini- lar portions of Dsg3 and BP180, components of desmo- cal presentation of the disease. Generally, it can be dif- somal and hemidesmosomal adhesion complexes of ferentiated into the mechanobullous or classic form of human skin, respectively. Dsg3- and BP180-reactive T EBA and inflammatory variants, resembling BP, Brun- cells produce T-helper 2 (Th2) cytokines, such as IL-4, 72 8 Treating Autoimmune Bullous Skin Disorders with Biologics

Fig. 8.3. Schematic overview of the immune pathogenesis of pemphigus vulgaris. Pem- phigusvulgaris(PV)isthe prototype of an autoantibody-mediated immunobul- lous skin disorder and is characterized by a loss of intraepidermal adhesion primarily caused by IgG autoantibodies specific for desmoglein (Dsg)3 and Dsg1, components of the desmosomal adhesion complex of epidermal keratinocytes that areconnectedtothekeratin cytoskeleton through interaction with the intracellular plaque proteins plakoglobin (PG) and desmoplakin (DP) (inset). IgG production by theautoreactiveBcellsis presumably regulated by Dsg3- and Dsg1-reactive Th1 and Th2 cells

IL-5 and IL-13, and presumably foster the production face of the CD20+ target B cells. Among several mecha- of autoantibodies of the Th2-dependent IgG4 subtype nisms involved in B-cell killing by rituximab, its B-cell which are preferentially seen in active stages of these cytolytic activity mainly depends on antibody-depen- disorders (Fig. 8.3). dent cell-mediated cytotoxicity (ADCC). Polymor- phisms of the Fc * RIII receptor which mediates ADCC seem to influence the response to rituximab. Other 8.2 mechanisms such as complement-mediated lysis and Rituximab (Anti-CD20 Monoclonal Antibody) induction of apoptosis may also contribute to the B- in the Treatment of Autoimmune Bullous cell-depleting activity of rituximab. Interestingly, there Skin Disorders is recent evidence suggesting that the mechanism of B- cell killing by rituximab depends on the tissue micro- 8.2.1 environment (Fig. 8.4). Biological Activity of Rituximab In vivo studies using hCD20 BAC (bacterial artificial Rituximab is a chimeric human/mouse IgG1 monoclo- chromosome) transgenic mice demonstrate that deple- nal antibody with human constant regions and variable tion of B lymphocytes varies among different B-cell murine regions derived from a murine anti-CD20 anti- compartments. In these mice, over 90% of the circulat- body (IDEC 2B8). Rituximab is directed toward CD20, ing B cells in the peripheral blood are depleted within a pan B-cell glycoprotein which is expressed on B lym- minutes. In lymph nodes and the spleen, B-cell deple- phocytes from the pre-B-cell stage to the pre-plasma- tion occurs within 24 h after application of rituximab cell stage. CD20 is a four-transmembrane glycoprotein and within 7 days in the peritoneal cavity where signifi- that is involved in B-cell differentiation and activation, cant B-cell depletion is delayed. Even though more than although its exact physiological function is not yet fully 90% of follicular B cells in the spleen are depleted with- understood. Several characteristics make the CD20 in 2 days after rituximab application, germinal center B antigen attractive for immunotherapy; since CD20 cells, particularly marginal zone B cells, appear to be does not circulate in the plasma, it is neither internal- more resistant to killing. These differences in resistance ized nor downregulated and there is no evidence that to rituximab are neither due to lack of CD20 expression antibody binding leads to shedding from the cell sur- nor due to differences in drug bioavailability. Thus, the 8.2 Rituximab (Anti-CD20 Monoclonal Antibody) in the Treatment of Autoimmune Bullous Skin Disorders 73

The current application of rituximab in autoantibody mediated autoimmune diseases was catalyzed by find- ings that treatment of B-cell NHL improved symptoms of lymphoma-associated autoimmune phenomena, since malignant B-cell clones are capable of secreting low-affinity self-reactive antibodies, for example autoantibodies specific for self antigens on red blood cells leading to autoimmune hemolytic anemia (Boye et al. 2003). Moreover, the rationale for applying rituximab in primary autoimmune disorders is the long-term depletion of pathogenic, autoantibody-secreting B-cell clones, thus restoring tolerance. Follow-up of rituximab-induced B-cell depletion in 24 patients with active RA showed that B-cell repopulation in the peripheral blood occurred around 8 months after rituximab treatment. Increased numbers of na¨ıve and immature B cells (CD19+, IgD+, CD38high,CD10low,CD24high)were identified during repopulation in the peripheral blood Fig. 8.4. Mode of action of rituximab. B-cell killing is largely similar to the B-cell populations found following bone- mediated by antibody-dependent cell mediated cytotoxicity marrow transplantation. In contrast, patients who (ADCC). Recent data indicates that the patient’s Fc * RIII allo- experienced a relapse of RA tended to show higher type seems to influence the response to rituximab. In vitro numbers of memory B cells during repopulation studies have shown that rituximab effectively induces comple- (Edwards et al. 2006). Another report by Rouziere et al. ment-dependent cytotoxicity (CDC) against B-cell lines and lymphoma cells. Induction of apoptosis may also contribute to (2005) demonstrated changes in the immunoglobulin B-cell killing heavy-chain repertoire after rituximab treatment, suggesting that new clones of B cells emerged from the differences in kinetics and sensitivity to the B-cell cyto- bone-marrow dominating the repopulation process. In lytic activity of rituximab seem to depend on protective most clinical studies treating autoimmune disorders, microenvironmental factors. The expression of the the oncological regimen consisting of four weekly i.v. CD20 antigen is not restricted to B cells. A small num- doses of 375 mg/m2 rituximab is adopted. However, dif- ber of T cells and NK cells also express low levels of ferent treatment protocols are reported with two single CD20. A recent study with 24 rheumatoid arthritis infusions, multiple courses including four weekly infu- (RA) patients showed that these cell populations disap- sions or single doses with up to 1 g rituximab. Besides pear for a mean of 5 months after rituximab therapy. its use in RA, rituximab is being investigated in numer- The authors did not find significant changes in the total ous autoimmune disorders including idiopathic throm- number or frequency of other T-cell subpopulations in bocytopenic purpura, autoimmune hemolytic anemia, the peripheral blood. lupus erythematosus, myasthenia gravis, Sjögren’s syn- dromeand,finally,autoimmunebullousdisorders. Within the latter group of disorders, mainly severe 8.2.2 cases of paraneoplastic pemphigus, pemphigus vulga- Clinical Experience with Rituximab Therapy ris, pemphigus foliaceus and, to a lesser extent, epider- In December 1997 the US Food and Drug Administra- molysis bullosa acquisita have been successfully treat- tion (FDA) approved rituximab for the treatment of ed with rituximab. relapsing or refractory indolent CD20+ B-cell non- Hodgkin’s lymphoma (NHL). More recently, rituximab 8.2.3 was also approved for the treatment of aggressive NHL Rituximab in Pemphigus in combination with standard chemotherapy in the US and in Europe. The use of rituximab in the treatment of There is a body of evidence demonstrating that rituxi- RA was endorsed by the FDA approval in March 2006. mab treatment is highly beneficial in the treatment of 74 8 Treating Autoimmune Bullous Skin Disorders with Biologics

recalcitrant clinical variants of pemphigus. As reported recently discussed for RA by Edwards and Cambridge for lymphoma patients and other autoimmune dis- (2006), the two-way interaction between autoreactive eases, four i.v. treatments with rituximab at 375 mg/m2 B-andT-cellclonesseemstobecrucialforboththeini- over 4 weeks induced a complete and sustained B-cell tiation and perpetuation of these autoantibody driven depletion in the peripheral blood which usually lasted autoimmune diseases. B cells have been shown to acti- for 6–9 months. As recently reviewed, all clinical stud- vate T cells through antigen presentation, and CD4+ ies and case reports using rituximab in severe recalci- Tcellsprovide“help”toBcellsthroughthedeliveryof trant pemphigus demonstrated good clinical responses cytokines and cell surface ligands, thus leading to a pos- with remissions lasting from several months to 2 years. itive feedback-loop. First evidence for this immunologi- A recent open-label study including one PF and four PV calhypothesisinpemphigusisprovidedbyarecent patients with therapy resistant disease showed that study investigating the frequency of Dsg3-specific auto- clinical improvement correlated with persistent B-cell reactive CD4+ T cells in the peripheral blood of PV depletion. However, clinical remission was not accom- patients undergoing rituximab therapy. In a group of panied by a significant decrease of desmoglein (Dsg)- 11 PV patients receiving the standard dose of rituximab, specific autoantibodies in all the patients (Arin et al. the frequencies of Dsg3-reactive CD4+ T cells were 2005). Another ongoing clinical study including a total determined by MACS cytokine secretion assay. Fre- of ten PV patients receiving the standard dose (four quencies of IL-4- (Th2 cells), IFN * -(Th1cells)andIL- weekly doses of 375 mg/m2) demonstrated a decrease of 10-producingtype1regulatoryTcells(Tr1Tcells)were Dsg1-/Dsg3-specific autoantibodies by 70–80% of the determined before and up to 12 months after rituximab initial titers within 2–3 months after rituximab treat- therapy. In all patients the frequencies of Dsg3-reactive ment and a further decrease by 40–50% of the initial CD4+ T cells decreased significantly compared to the autoantibody levels 6 months after therapy, correlating pre-treatment values. The frequencies of tetanus tox- with a good clinical response (Fig. 8.5). In contrast, oid-specific T cells in these patients were not affected by only minor changes were detected in anti-tetanus tox- B-cell depletion and showed only minor changes in fre- oid IgG antibodies, suggesting that autoreactive B cells quency. These results suggest that activation of autore- and their corresponding plasma cells tended to have active T cells in pemphigus largely depend on continu- short life spans whereas allo-reactive plasma cells (as ous antigen presentation by autoreactive B cells deliver- for post-vaccination immunity) were more resistant to ing activation signals. rituximab and had longer life spans. Compared to other autoimmune diseases such as RA 8.2.4 and systemic lupus erythematosus, in pemphigus as an Rituximab in Epidermolysis Bullosa Acquisita autoantibody mediated autoimmune disorder, the major autoantigens (Dsg1 and Dsg3), pathogenic auto- A recent case report described an inflammatory variant antibodies and autoreactive, Dsg3-specific CD4+ T cells of EBA with lesions on the trunk, the hands and the oral have been identified and functionally characterized. As mucosa. The patient received four courses of rituximab

Fig. 8.5. Clinical response in a patient with mucosal pemphigus vulgaris (PV) to treatment with rituximab. The PV patient with extensive oral erosions shown here was refractory to immunosuppressive treatment including mycophenolate mofetil (3 g/day) and azathioprine (1.75 mg/kg/ day), respectively, in combination with methylpredniso- lone (0.5–1.5 mg/kg/day) for 6months(A, before rituximab treatment). Rituximab treatment led to a rapid clinical response (B, 2 months after rituximab treatment) 8.2 Rituximab (Anti-CD20 Monoclonal Antibody) in the Treatment of Autoimmune Bullous Skin Disorders 75

Fig. 8.6. Clinical response of mechanobullous epidermolysis bullosa acquisita to rituximab. Patient 1 showed blisters and erosions on his hands (a, left), feet and the oral mucosa before rituximab treatment. Marked improvement of the lesions on the hands was noted 15 weeks after completion of therapy (a, right), while erosions of the oral mucosa remained largely unaffected. Patient 2 (b, left)showed extensive blisters and erosions of the trunk and hands. Nine months after completion of rituximab therapy, an almost complete clinical remission with post- inflammatory atrophic hyperpigmentations on the chest and a few crusty erosions was visible (b, right)

(i.v., 375 mg/m2)asanadjuvanttreatmentinweekly with rituximab 375 mg/m2 ×4 over a period of 4 weeks. intervals. Seven weeks after the completion of rituxi- Concomitant medication with mycophenolate mofetil mab therapy, the patient was in complete remission 3 g/day was maintained during and after B-cell deple- and received tapering doses of azathioprine (175 mg/ tion therapy. Within 15 weeks gradual improvement of day), colchicine (160 mg/day) and prednisolone the lesions on the hands was perceived, whereas the (80 mg/day). As an unusual adverse event a deep oral lesions and the lesions on the soles showed only venous thrombosis of the lower leg developed between slight improvement (Fig. 8.6). A second patient with the first and second infusion of rituximab. mechanobullous EBA refractory to treatment to a vari- In contrast, administration of rituximab in patients ety of systemic immunosuppressive drugs in the past, with mechanobullous EBA shows a different clinical who had lesions that mainly affected the trunk and the response. In our own experience, an EBA patient with hands,wastreatedinthesamewayastheprevious erosive lesions of the oral mucosa, the esophagus and patient with rituximab. Within 8 months his disease the nasopharynx and tense blisters on the hands, lower was well controlled on monotherapy with rituximab legsandfeetshowedonlyapartialclinicalresponseto with only very few residual crusty erosions and atro- rituximab. Subsequently, he received adjuvant therapy phic lesions. The excellent clinical response was accom- 76 8 Treating Autoimmune Bullous Skin Disorders with Biologics

panied by a decline in titres of circulating autoanti- reduced immunogenicity, the presence of human antibodies, which were undetectable & 9monthsaftercom- chimeric antibodies (HACA) is a rare event which has pletion of rituximab therapy. Complete B-cell depletion been reported in less than 1% of treated patients with persisted for & 9 months. In both patients rituximab NHL. In patients with HACA impairing the function of was well tolerated with no side effects. rituximab, a newly designed humanized monoclonal anti-CD20antibody(hCD20)mightbeeffective. 8.2.5 Toxicity of Rituximab Treatment and Adverse Effects 8.2.6 Contraindications for Treatment with Rituximab In general, rituximab is well tolerated in both patients with malignancies and those with autoimmune bullous Ongoing or recurrent infectious disorders including disorders. Except for two reports of serious infections clinically apparent bacterial or viral infections exclude including septic arthritis, Pneumocystis carinii pneu- the application of rituximab. Patients with a history of monia and pneumonia after treatment of pemphigus malignancies or immunodeficiency syndromes, as well patients with rituximab, the most common adverse as pregnant or lactating women, should not undergo events are infusion-related events occurring during or rituximab therapy. Patients with cardiac disorders shortly after the first infusion. Larger studies with lym- should be closely monitored during therapy with ritu- phoma patients demonstrated that about 95% of the ximab. A history of anaphylactic hypersensitivity reac- infusion-related symptoms such as chills, fever, head- tions against mouse proteins also excludes treatment ache, rhinitis, pruritus or vasodilatation were mild to with rituximab. moderate. Most of these symptoms could be avoided by pre-treatment with antihistamines, antipyretics and steroids. Another report showed that a severe cytokine 8.3 release syndrome more frequently occurred in lym- Inhibitors of TNF-␣ in the Treatment of phoma patients with a higher tumor burden in their Autoimmune Bullous Skin Disorders peripheral blood which might be correlated with 8.3.1 increased serum levels of tumor necrosis factor-alpha Central Role of TNF-␣ in Inflammation (TNF- [ ) and interleukin 6 (IL-6) after infusion of rituximab. At present, severe cytokine release syndromes Tumor necrosis factor- [ is a proinflammatory cytokine have not yet been reported in patients with bullous which plays a key role in most of the inflammatory pro- autoimmune disorders. Hematological toxicity has cesses as well as in immune responses to infections and been reported in about 10% of patients including a tumor antigens. TNF- [ is largely produced and temporary reduction of platelets or neutrophils. released by macrophages and monocytes. It exists as a Despite the depletion of B cells for several months the soluble 17-kDa sized protein made of three subunits. risk for infectious diseases does not seem to be signifi- The cytokine stands at the beginning of a cascade of cantly higher in patients treated with rituximab. A pro-inflammatory cytokines and triggers off pro- recent study in RA patients demonstrated a significant inflammatory signals at the target cell by binding to decrease of serum rheumatoid factor (IgM, IgG and IgA membrane-based TNF receptors, e.g., on T cells and isotypes) over several months, whereas total immuno- macrophages. Furthermore, TNF- [ causes a rise in globulinlevelsdecreasedbyonly25%forIgGandIgA body temperature as well as the induction of the acute and by 43% for IgM and the titers for pneumococcal phase proteins, an increased migration of dendritic polysaccharide-reactive antibodies remained un- cells from the periphery to regional lymph nodes and changed. Another recent report showed unaffected lev- an activation of neutrophils. In inflammatory diseases, elsofanti-tetanustoxoidantibodiesinpatientstreated TNF- [ possesses pleiotropic effects and leads, among with rituximab. These observations help to explain other factors, through the activation of a pro-inflam- why, despite complete depletion of B cells for several matory cytokine cascade, to an induction of adhesion months, the risk for secondary infections does not molecules on endothelial cells (E-selectin, ICAM-1), seem to be significantly higher in patients treated with which leads to an increased migration of leukocytes. rituximab. As rituximab is a chimeric antibody with Furthermore, TNF- [ induces the secretion of metallo- 8.3 Inhibitors of TNF-␣ in the Treatment of Autoimmune Bullous Skin Disorders 77 proteinases and the release of pro-inflammatory cyto- constant region (Fc)ofhumanIgG1.Infliximabbinds kines(IL-1,IL-6,IL-8,GM-CSF).TNF-[ -conveyed with high specificity and affinity to free and mem- induction of pro-inflammatory cytokines, leukocyte brane-bound TNF- [ , which is expressed at the surface chemotaxis and angiogenesis possibly play a funda- by activated T cells and macrophages. Besides the mental role in autoimmune diseases of the skin, pre- blocking of TNF- [ , the lysis of the target cell may also sumably diseases which are characterized by elevated occur through activation of complement. This proba- TNF- [ serum concentrations, fever and an increase of bly accounts for the antibody conveying a cytotoxic acute phase proteins. Elevated serum levels of TNF- [ effect of infliximab. Inhibition of the pro-inflammatory aredetectableinmanyautoimmunediseasesincluding effects of TNF- [ results through the formation of stable RA, psoriasis and Crohn’s disease. complexes. In addition, the migration of leukocytes and the release of TNF- [ -dependent proinflammatory cytokines like IL-1 and IL-6 is inhibited. 8.3.2 Adalimumab is a human monoclonal IgG antibody Inhibition of TNF-␣ by Biologics 1 containing only human peptide sequences. It binds AnewclassofTNF-[ inhibitors are the so-called “bio- with high specificity and affinity to soluble and mem- logics,” which are either recombinant monoclonal anti- brane-bound TNF- [ and blocks its interaction with the bodies or soluble TNF receptor fusion proteins. These p55 and p75 cell surface TNF receptors, thereby neu- proteins can be either isolated from animal tissues or tralizing the biological activities of this cytokine. Ada- commonly synthesized by biotechnological methods. limumab also modulates TNF-induced or regulated Themoredefinedunderstandingofthepathophysiolo- biological responses, such as changes in the levels of gy of autoimmune diseases has led to the therapeutic adhesion molecules responsible for leukocyte migra- use of biologics in chronic inflammatory disorders tion. (Scheinfeld 2004). At present, a few uncontrolled case Etanercept is a recombinant human fusion protein reports suggest that the therapeutic blockade of TNF- [ whichconsistsoftwosolublep75TNFreceptorsand may be a novel option for the short-term control of oth- the Fc portion of human IgG1. Etanercept possesses a erwise recalcitrant autoimmune bullous skin disorders dimeric structure with high affinity to TNF- [ ,andthe (Table 8.1). linkage to the Fc portion of human IgG produces a lon- Infliximab is a chimeric monoclonal antibody con- ger half-life. Etanercept neutralizes TNF- [ better than [ sisting of a murine anti-TNF- Fab fragment and the the monomeric soluble p75 receptor. By blocking the

Table 8.1. TNF- [ inhibitors and application in autoimmune bullous dermatoses Infliximab (Remicade␤) Adalimumab (Humira␤)Etanercept(Enbrel␤) Molecule Chimeric (murine-human) Fully human monoclonal Humanreceptorfusionprotein monoclonal antibody (IgG1) IgG1 antibody (p75-TNF-receptor dimer and IgG1) Target structure Free and membrane-bound Free and membrane-bound Free TNF- [ and lymphotoxin- [ TNF- [ TNF- [ (TNF- q ) Dosage 3–5 mg/kg/day intravenous 40 mg every other week 25 mg (children 0.4 mg/kg/day) infusion at weeks 0, 2 and 6; subcutaneously twice weekly subcutaneously; then every 8 weeks alternative 50 mg twice weekly at first 12 weeks Efficacy in autoimmune Pemphigus vulgaris IgA pemphigus Pemphigus vulgaris bullous skin disorders Jacobi et al. 2005 Howell et al. 2005 Berookhim et al. 2004 Pardo et al. 2005 Lin et al. 2005 Mucous membrane pemphigoid Sacher et al. 2002 Bullous Pemphigoid Yamauchi et al. 2006 78 8 Treating Autoimmune Bullous Skin Disorders with Biologics

circulating TNF- [ trimers the interaction of TNF- [ 6weeksatweeks0,2and6.Leflunomide(20mg/day) with the cell bound receptors is prevented and the pro- and prednisone (0.25 mg/kg/day) were continued dur- inflammatory cytokine cascade interrupted. Etaner- ing the infliximab regimen as adjuvant treatment and cept,inaddition,bindslymphotoxin(TNF-q ). Cells with the aim of preventing formation of anti-infliximab with membrane-bound TNF- [ bind etanercept; lysis antibodies (HACA). Within 48 h of the first dose of due to ADCC of these cells does not occur in contrast to infliximab, blister formation ceased, and within the the binding of infliximab (Table 8.1). first4dayscutaneousblistersandmucosalerosions gradually disappeared (Fig. 8.7). Infliximab infusions were discontinued 8 weeks after initiation when the 8.3.3 patient developed varicella zoster of the left S1/2 der- Inhibition of TNF-␣ in Pemphigus Vulgaris matome, which was treated successfully with i.v. acy- Inpemphigusvulgaris(PV),autoantibodiesagainst clovir. Of note, the patient’s clinical status remained Dsg1 and Dsg3 have been shown to induce loss of kera- stable with few cutaneous erosions during the previ- tinocyte adhesion upon binding to desmosomal target ously insufficient therapy with leflunomide (20 mg/d) autoantigens leading to the release of TNF- [ and IL-1 and prednisone (0.25 mg/kg/day). After a relapse from epidermal keratinocytes, which presumably 4 months later the disease was controlled by several enhance the process of blister formation. In vivo, an cycles of immunoadsorption therapy in combination increased expression of TNF- [ and IL-1 is found in the with mycophenolate mofetil (3 g/day). direct environment of the intra-epidermal loss of adhe- Further case reports confirm the successful manage- sion. In vitro, cultures of human keratinocytes are ment of severe pemphigus with infliximab. A 62-year- resistant to the acantholytic effect of pathogenic, Dsg3- old male with PV and generalized bullous lesions and a specific autoantibodies upon pre-treatment with anti- severe oral stomatitis was managed with systemic cor- TNF- [ antibodies. This protective effect of blocking ticosteroids for 6 years, mycophenolate mofetil, cyclo- TNF- [ can be reproduced in vivo. Feliciani et al. (2000) sporin A, azathioprine, cyclophosphamide, methotrex- showed that the injection of anti-Dsg3 IgG in TNF ate, thalidomide and plasmapheresis without much receptor 1/2 deficient mice led more rarely to blister success. As a last resort, the patient received infliximab formation than the injection of the identical autoanti- (5mg/kg/di.v.)at0,2and6weeksandthenevery bodies in syngeneic “wild type” mice with an intact 8 weeks. No other drugs were needed as concomitant TNF receptor. medication. The patient improved dramatically after Based on these observations, the potent TNF- [ five infusions leading to total clearance of active lesions inhibitor, infliximab, was used in a 43-year-old male at week 22 without severe side effects. After the 13th with a therapy refractory PV with a history of pulmo- infusion, treatment with infliximab discontinued nary embolism, arterial hypertension, steroid-induced because new cutaneous and oral lesions had appeared. osteoporosis and diabetes mellitus who presented with The disease was eventually controlled with oral corti- disseminated cutaneous blisters and extensive erosions costeroids and rituximab. of the oral mucosa (Jacobi et al. 2005). The patient was There is a report on the effective treatment of PV given three doses of infliximab (5 mg/kg/day i.v.) over with the TNF- [ antagonist, etanercept, in a 62-year-old

Fig. 8.7. Immediate clinical response of pemphigus vulgaris (PV) to treatment with the TNF inhibitor infliximab. Cutaneous bullae in a patient with recalcitrant PV before (A)and2daysafter(B)the first infliximab infusion 8.4 Summary 79 woman with long-standing cutaneous PV and concom- maintained for more than 8 months with low-dose itant seronegative arthritis. Upon initiation of treat- prednisone treatment (1 mg/day). ment with etanercept for arthritis, the cutaneous blis- There is also a report about the treatment of coexist- ters gradually disappeared. Lin et al. (2005) reported ing BP and psoriasis with the TNF- [ antagonist, eta- the successful treatment of recalcitrant PV and pem- nercept. A 64-year-old man with a long history of phigus vegetans with etanercept and carbon dioxide plaque-type psoriasis developed acute symptoms of BP. laser. Initial therapy consisted of mycophenolate mofetil (2 g/ The human anti-TNF- [ monoclonal antibody, adali- day), which had no effect on the clinical activity of BP. mumab, has in combination with mycophenolate mofe- Mycophenolate mofetil was discontinued, and he was tilbeensuccessfullyemployedinthesubcornealpustu- startedonprednisone(60mg/day).Thecutaneousbul- lar dermatosis subtype of IgA pemphigus. A 41-year- lae disappeared almost completely by the 10th day and old male with IgA pemphigus failed multiple treat- also the plaque psoriasis improved. Toprevent rebound ments, including acitretin, broadband ultraviolet B of psoriasis and BP during the tapering phase of pred- therapy, dapsone, methotrexate and oral steroids. He nisone, etanercept (50 mg s.c. weekly) was added to the showed improvement to alefacept, but treatment was treatment regimen. At a dose of 20 mg/day prednisone, discontinued because of a low peripheral CD4+ T-cell new cutaneous blisters developed. Etanercept was count. Cyclosporine was also effective, but was discon- increasedto50mgtwiceweeklyandtheblisterssubse- tinued secondary to elevated creatinine levels. Adali- quently resolved. When prednisone treatment was mumab (40 mg s.c. every other week) was started, and eventually discontinued, the clinical activity of BP after the third dose his skin completely cleared. Myco- remained silent; no adverse events occurred. phenolate mofetil (1 g/day) was used as concomitant medication. The patient has been symptom-free for 5 months, besides the occasional occurrence of a few 8.4 pustules. Summary

Rituximab has clearly evolved as a novel therapeutic 8.3.4 option in refractory autoimmune bullous skin disor- Inhibition of TNF-␣ in Bullous Pemphigoid ders such as severe pemphigus vulgaris and epidermo- There are a few reports on the effect of TNF- [ antago- lysis bullosa acquisita. The mode of action suggests nistsinclinicalvariantsofBP,anautoimmunebullous that not only the production of autoantibodies is inhib- skin disorder associated with IgG autoantibodies ited by depletion of autoreactive B cells but also the against components of the dermo-epidermal basement critical interaction of autoreactive T and B cells, which membrane zone, such as BP180 and BP230. In the clini- is essential for the perpetuation of the ongoing autoim- cal variant mucous membrane pemphigoid, a disorder mune response. Treatment of pemphigus with rituxi- which is characterized by chronic blistering of the mab has been shown to be highly effective in refractory mucous membranes with secondary scarring, the TNF- mucocutaneous pemphigus with a prolonged biologi- [ blocker etanercept has been applied with great suc- cal activity due to the long-term depletion of autoreac- cess. A 72-year-old woman with long-standing mucous tive B cells and a lack of major side effects. In addition, membrane pemphigoid and acute exacerbation of oral single case reports suggest that TNF- [ blockers may be lesions had already been treated with prednisone a therapeutic option in pemphigus and mucous mem- (1 mg/kg/day) in combination with azathioprine brane pemphigoid with life-threatening or therapy (100 mg/day) and mycophenolate mofetil (2×1 g/day) refractory disease course, particularly as a short-term over a year, leading to a moderate clinical response. intervention therapy. However, potential side effects Etanercept (25 mg s.c. ×2/week) in combination with due to the immunosuppressive potential of TNF- [ prednisone (initially 60 mg/day) led after the third antagonists such as severe bacterial infections, viral cycle to the disappearance of newly developed blisters. infections and tuberculosis should be carefully consid- Even though the corticosteroids were gradually ered and constitute a serious hazard in patients on tapered, the patient was symptom-free after a total of chronic immunosuppressive therapy. six etanercept injections and clinical remission was 80 8 Treating Autoimmune Bullous Skin Disorders with Biologics

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