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Rituximab decreases without preference all subclasses of IgG anti-BP180 autoantibodies in refractory bullous pemphigoid (BP)
Journal of Dermatological Science 74 (2014) 93–94
Contents lists available at ScienceDirect
Journal of Dermatological Science journal homepage: www.jdsjournal.com
Letter to the Editor Rituximab decreases without preference all subclasses of IgG anti-BP180 autoantibodies in refractory bullous pemphigoid (BP)
Keywords: Bullous pemphigoid Auto-antibodies: IgG subclass Rituximab
Bullous pemphigoid (BP) is characterized by autoantibodies targeting the immunodominant non-collagenous 16A domain (NC16A) of the BP180 antigen (type XVII collagen) with circulating titers correlating with disease severity [1]. IgG subclass analysis demonstrates the predominance of IgG1 and IgG4 anti-BP180 autoantibodies that are believed to correlate with disease severity or duration [2–5]. To a lesser extent IgG2 and IgG3 anti-BP180 autoantibodies are also present and in some cases have been described as pathogenic [5]. Rituximab targets the B cell specific surface marker CD20 and has been used successfully to treat a variety of autoimmune diseases including autoimmune blistering diseases [6,7]. Clinical improvement in patients with pemphigus has been associated with decreased levels of circulating auto-antibodies [7]. Some patients however do not experience total remissions and variable levels of autoantibody may persist after rituximab therapy [8]. Recently, Khosroshahi et al. reported clinical improvement in 9 of 10 subjects with IgG4-related disease after rituximab treatment [9]. All 10 subjects were able to discontinue steroid and DMARD therapy, which was associated with a decrease in only the presumed pathogenic serum IgG4 antibodies. These observations suggest that rituximab may have a preferential effect on IgG4 autoantibodies with resultant clinical improvement. Since both IgG1 and IgG4 anti-NC16A BP180 autoantibodies have been suggested to be pathogenic in BP, an explanation for the variability in the rituximab induced clinical remission in BP could
be a preferential decrease of the IgG4 anti-NC16A BP180 autoantibodies with a persistence of pathogenic IgG1 autoantibodies. The purpose of this study is to investigate the change in serum levels of anti-NC16A BP180 total IgG and IgG1–G4 autoantibodies after rituximab treatment. Sera were collected from six refractory BP subjects before treatment with rituximab as a part of an open label, prospective study [10]. All subjects had circulating IgG anti BP 180 antibodies. Sera were collected before therapy (T0) and at, 0.5, 1–6, 9, and 12 months after rituximab treatment (1000 mg infusion of rituximab, days 0 and 15). Sera from 20 controls (ten healthy; five pemphigus vulgaris (PV); five pemphigus foliaceus (PF)) were analyzed. This study was approved by the institutional review board at Duke University Medical Center. Two subjects (subjects 5 and 6, Table 1) experienced a disease flare at months 11.5 and 7 respectively. The remaining subjects were in remission at 12 months [10]. Serum anti-NC16A BP180 total IgG autoantibodies were defined as positive if values exceeded 9 IU of anti-NC16A BP180 total IgG by ELISA (MBL Int. Corp., Woburn, MA). Sera were diluted to insure that all values were in the linear range of the assay and values calculated based on the dilution factor. NC16A BP180 ELISA plates (MBL Int. Corp., Woburn, MA) were utilized for detection of serum IgG1–G4 anti-NC16A BP180 autoantibodies. First, a single positive BP subject serum with an OD value greater than 0.9 was selected as a positive control for each IgG subclass ELISA assay. IU were calculated as described for the total IgG ELISA using the single BP subject positive control serum. Positivity for the IgG1–G4 isotypes binding was defined as any value exceeding 3 times the highest value of the 10 healthy subject control sera (Table 1). All BP subjects had IgG anti-NC16A BP180 antibodies at T0, while, 5/6 were positive for IgG1, 5/6 for IgG2, 4/6 for IgG4, and 2/6 for the IgG3 isotype (Table 1). IgG anti-NC16A BP180 autoantibodies were not detected in the skin disease control subjects (1.6 IU for PF (N = 5) and 1.4 IU for PV (N = 5)). The IgG1–G4 subclass anti-NC16A BP 180 antibodies were negative in all of the skin disease control subjects. The total IgG anti-NC16A BP180 autoantibodies of the six refractory BP subjects decreased to a mean nadir of 23.4 IU (nadir
Table 1 Total IgG and IgG1–G4 anti-NC16A BP180 autoantibodies before rituximab treatment (T0). ‘‘Total positive’’ represents number of subject studied with index units (IU) exceeding 9 IU for total IgG or exceeding 3 the highest control for the IgG1–G4 anti-NC16A BP180 autoantibodies over total subjects tested. Subject number
Total IgG index units anti-NC16A BP180
IgG1 index units anti-NC16A BP180
IgG2 index units anti-NC16A BP180
IgG3 index units anti-NC16A BP180
IgG4 index units anti-NC16A BP180
Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Total positive (%) Normal
64 37 606 137 40 3556 6/6 (100%) 9
12.5 22.6 384.1 19.6 2.1 237.5 5/6 (83%) 6.9
127.4 1 92 178.7 8.7 6152.4 5/6 (83%) 2.4
4.3 2.6 4.1 4.2 45.6 12.3 2/6 (33%) 5.4
950 0.9 29.2 48.0 0.4 830.6 4/6 (67%) 1.8
0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.11.014
Letter to the Editor / Journal of Dermatological Science 74 (2014) 93–94
94
IgG Anti-NC16A BP180 After Rituximab 45 3000
Total IgG (N=6)
40
IgG1 (N=5)
35
IgG3 (N=2)
30
IgG4 (N=4)
25
Prednisone (N=6)
1500
20 15
1000
10 500
mg / day
Index Units + SEM
2000
IgG2 (N=5)
Prednisone
2500
IgG autoantibodies. Furthermore, the time to the lowest antibody level did not differ between the different IgG subclass nor when compared to total IgG anti-NC16A BP180. These findings suggest that any difference in the therapeutic effect of rituximab in the treatment of patients with BP is not due to a preferential decrease of IgG4 autoantibodies. Funding support Research grant: Genentech Inc. (RP Hall III, PI). References
5
0
0 0 0.5 1
2
3
4
5
6
9 12
Months Fig. 1. Mean + SEM (standard error of the mean) total IgG, IgG1–G4 anti-NC16A BP180 autoantibodies, and prednisone (mg/day) treatment dose decrease after rituximab therapy in refractory BP. Rituximab decreases anti-NC16A BP180 IgG1– G4 autoantibodies without IgG subclass preference. No appreciable differences were found in the time to reach the nadir for the IgG1–G4 as compared to total IgG anti-NC16A BP180 autoantibodies (WRS, NS). Autoantibody levels are indicated in IU.
range of 5–53.6 IU; mean 28.5% of T0; Wilcoxon rank sum (WRS), p < 0.05 compared to T0) at a mean of 9.5 months (range, 6–12 months). The IgG1 anti-NC16A BP180 autoantibodies decreased to a mean nadir of 3.9 IU (mean 13.6% of T0; range of 1.7–6.4 IU; WSR p value < 0.05 compared to T0) at a mean of 10.2 months (range 6–12 months), while IgG2 anti-NC16A BP180 autoantibodies decreased to a mean nadir of 25.2 IU (mean 23.4% of T0, range 0–86.2 IU; WSR p value < 0.05) at a mean 8.2 months (range 5–12 months). The 2 subjects with detectable anti-NC16A BP180 IgG3 decreased to a mean nadir of 1.5 IU (mean decrease 3.2% of T0) at 3 months. Four subjects were found to have detectable IgG4 antiNC16A autoantibodies that decreased to a mean nadir 4.3% of T0, range 0.2–115 (WSR; p value = 0.063) at 8.3 months however the lowest values did not reach a statistical significance (p = .0625, WRS). No significant difference was seen between the % decrease from T0 in total IgG and the decrease for the other IgG subclasses. Similarly, analysis of the time at which the nadir value occurred revealed, no difference between the time to the nadir for total IgG and the IgG subclass autoantibodies (Fig. 1). There was no difference noted in the change of total IgG or IgG subclass reduction between the 5 subjects with no disease flare and the 2 with a disease flare. The subjects who flared however did have an increase in the level of IgG anti NC16A BP 180 at the time of the flare [10]. Analysis of the levels of IgG anti NC16A domain BP180 specific autoantibodies in subjects with BP for one year after rituximab therapy revealed that treatment did not preferentially decrease any specific IgG subclass of autoantibodies as compared to the total
[1] Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Liu Z, Diaz LA. Bullous pemphigoid and herpes gestationis autoantibodies recognize a common noncollagenous site on the BP180 ectodomain. J Immunol 1993;151:5742–50. [2] Zillikens D, Mascaro JM, Rose PA, Liu Z, Ewing SM, Caux F, et al. A highly sensitive enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid. J Invest Dermatol 1997;109:679–83. [3] Schmidt E, Obe K, Brocker EB, Zillikens D. Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid. Arch Dermatol 2000;136:174–8. [4] Hofmann S, Thoma-Uszynski S, Hunziker T, Bernard P, Koebnick C, Stauber A, et al. Severity and phenotype of bullous pemphigoid relate to autoantibody profile against the NH2- and COOH-terminal regions of the BP180 ectodomain. J Invest Dermatol 2002;119:1065–73. [5] Laffitte E, Skaria M, Jaunin F, Tamm K, Saurat JH, Favre B, et al. Autoantibodies to the extracellular and intracellular domain of bullous pemphigoid 180, the putative key autoantigen in bullous pemphigoid, belong predominantly to the IgG1 and IgG4 subclasses. Br J Dermatol 2001;144:760–8. [6] Schmidt E, Seitz CS, Benoit S, Brocker EB, Goebeler M. Rituximab in autoimmune bullous diseases: mixed responses and adverse effects. Br J Dermatol 2007;156:352–6. [7] Joly P, Mouquet H, Roujeau JC, D’Incan M, Gilbert D, Jacquot S, et al. A single cycle of rituximab for the treatment of severe pemphigus. N Engl J Med 2007;357:545–52. [8] Kasperkiewicz M, Shimanovich I, Ludwig RJ, Rose C, Zillikens D, Schmidt E. Rituximab for treatment-refractory pemphigus and pemphigoid: a case series of 17 patients. J Am Acad Dermatol 2011;65:552–8. [9] Khosroshahi A, Carruthers M, Deshpande V, Unizony S, Bloch D, Stone J. Rituximab for the treatment of IgG4-related disease: lessons from 10 consecutive patients. Medicine (Baltimore) 2012;91:57–66. [10] Hall RP, Streilein RD, Hannah D, McNair P, Fairley JA, Ronaghy A, et al. Association of serum B cell activating factor (BAFF) level and proportion of memory and transitional B cells with clinical response after rituximab treatment of bullous pemphigoid patients. J Invest Dermatol 2013. http:// dx.doi.org/10.1038/jid.2013.236 [advanced online publication 27 June 2013].
Arash Ronaghy, Robert D. Streilein, Russell P. HallIII* Department of Dermatology, Duke University Medical Center, Durham, NC 27710, USA *Corresponding author at: Box 3135, Duke University Medical Center, Durham, NC 27710, USA. Tel.: +1 919 684 3110; fax: +1 919 684 3002 E-mail addresses: [email protected] [email protected] (R.P. Hall III). Received 3 July 2013 Accepted 28 November 2013
Contents lists available at ScienceDirect
Journal of Dermatological Science journal homepage: www.jdsjournal.com
Letter to the Editor Rituximab decreases without preference all subclasses of IgG anti-BP180 autoantibodies in refractory bullous pemphigoid (BP)
Keywords: Bullous pemphigoid Auto-antibodies: IgG subclass Rituximab
Bullous pemphigoid (BP) is characterized by autoantibodies targeting the immunodominant non-collagenous 16A domain (NC16A) of the BP180 antigen (type XVII collagen) with circulating titers correlating with disease severity [1]. IgG subclass analysis demonstrates the predominance of IgG1 and IgG4 anti-BP180 autoantibodies that are believed to correlate with disease severity or duration [2–5]. To a lesser extent IgG2 and IgG3 anti-BP180 autoantibodies are also present and in some cases have been described as pathogenic [5]. Rituximab targets the B cell specific surface marker CD20 and has been used successfully to treat a variety of autoimmune diseases including autoimmune blistering diseases [6,7]. Clinical improvement in patients with pemphigus has been associated with decreased levels of circulating auto-antibodies [7]. Some patients however do not experience total remissions and variable levels of autoantibody may persist after rituximab therapy [8]. Recently, Khosroshahi et al. reported clinical improvement in 9 of 10 subjects with IgG4-related disease after rituximab treatment [9]. All 10 subjects were able to discontinue steroid and DMARD therapy, which was associated with a decrease in only the presumed pathogenic serum IgG4 antibodies. These observations suggest that rituximab may have a preferential effect on IgG4 autoantibodies with resultant clinical improvement. Since both IgG1 and IgG4 anti-NC16A BP180 autoantibodies have been suggested to be pathogenic in BP, an explanation for the variability in the rituximab induced clinical remission in BP could
be a preferential decrease of the IgG4 anti-NC16A BP180 autoantibodies with a persistence of pathogenic IgG1 autoantibodies. The purpose of this study is to investigate the change in serum levels of anti-NC16A BP180 total IgG and IgG1–G4 autoantibodies after rituximab treatment. Sera were collected from six refractory BP subjects before treatment with rituximab as a part of an open label, prospective study [10]. All subjects had circulating IgG anti BP 180 antibodies. Sera were collected before therapy (T0) and at, 0.5, 1–6, 9, and 12 months after rituximab treatment (1000 mg infusion of rituximab, days 0 and 15). Sera from 20 controls (ten healthy; five pemphigus vulgaris (PV); five pemphigus foliaceus (PF)) were analyzed. This study was approved by the institutional review board at Duke University Medical Center. Two subjects (subjects 5 and 6, Table 1) experienced a disease flare at months 11.5 and 7 respectively. The remaining subjects were in remission at 12 months [10]. Serum anti-NC16A BP180 total IgG autoantibodies were defined as positive if values exceeded 9 IU of anti-NC16A BP180 total IgG by ELISA (MBL Int. Corp., Woburn, MA). Sera were diluted to insure that all values were in the linear range of the assay and values calculated based on the dilution factor. NC16A BP180 ELISA plates (MBL Int. Corp., Woburn, MA) were utilized for detection of serum IgG1–G4 anti-NC16A BP180 autoantibodies. First, a single positive BP subject serum with an OD value greater than 0.9 was selected as a positive control for each IgG subclass ELISA assay. IU were calculated as described for the total IgG ELISA using the single BP subject positive control serum. Positivity for the IgG1–G4 isotypes binding was defined as any value exceeding 3 times the highest value of the 10 healthy subject control sera (Table 1). All BP subjects had IgG anti-NC16A BP180 antibodies at T0, while, 5/6 were positive for IgG1, 5/6 for IgG2, 4/6 for IgG4, and 2/6 for the IgG3 isotype (Table 1). IgG anti-NC16A BP180 autoantibodies were not detected in the skin disease control subjects (1.6 IU for PF (N = 5) and 1.4 IU for PV (N = 5)). The IgG1–G4 subclass anti-NC16A BP 180 antibodies were negative in all of the skin disease control subjects. The total IgG anti-NC16A BP180 autoantibodies of the six refractory BP subjects decreased to a mean nadir of 23.4 IU (nadir
Table 1 Total IgG and IgG1–G4 anti-NC16A BP180 autoantibodies before rituximab treatment (T0). ‘‘Total positive’’ represents number of subject studied with index units (IU) exceeding 9 IU for total IgG or exceeding 3 the highest control for the IgG1–G4 anti-NC16A BP180 autoantibodies over total subjects tested. Subject number
Total IgG index units anti-NC16A BP180
IgG1 index units anti-NC16A BP180
IgG2 index units anti-NC16A BP180
IgG3 index units anti-NC16A BP180
IgG4 index units anti-NC16A BP180
Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Total positive (%) Normal
64 37 606 137 40 3556 6/6 (100%) 9
12.5 22.6 384.1 19.6 2.1 237.5 5/6 (83%) 6.9
127.4 1 92 178.7 8.7 6152.4 5/6 (83%) 2.4
4.3 2.6 4.1 4.2 45.6 12.3 2/6 (33%) 5.4
950 0.9 29.2 48.0 0.4 830.6 4/6 (67%) 1.8
0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.11.014
Letter to the Editor / Journal of Dermatological Science 74 (2014) 93–94
94
IgG Anti-NC16A BP180 After Rituximab 45 3000
Total IgG (N=6)
40
IgG1 (N=5)
35
IgG3 (N=2)
30
IgG4 (N=4)
25
Prednisone (N=6)
1500
20 15
1000
10 500
mg / day
Index Units + SEM
2000
IgG2 (N=5)
Prednisone
2500
IgG autoantibodies. Furthermore, the time to the lowest antibody level did not differ between the different IgG subclass nor when compared to total IgG anti-NC16A BP180. These findings suggest that any difference in the therapeutic effect of rituximab in the treatment of patients with BP is not due to a preferential decrease of IgG4 autoantibodies. Funding support Research grant: Genentech Inc. (RP Hall III, PI). References
5
0
0 0 0.5 1
2
3
4
5
6
9 12
Months Fig. 1. Mean + SEM (standard error of the mean) total IgG, IgG1–G4 anti-NC16A BP180 autoantibodies, and prednisone (mg/day) treatment dose decrease after rituximab therapy in refractory BP. Rituximab decreases anti-NC16A BP180 IgG1– G4 autoantibodies without IgG subclass preference. No appreciable differences were found in the time to reach the nadir for the IgG1–G4 as compared to total IgG anti-NC16A BP180 autoantibodies (WRS, NS). Autoantibody levels are indicated in IU.
range of 5–53.6 IU; mean 28.5% of T0; Wilcoxon rank sum (WRS), p < 0.05 compared to T0) at a mean of 9.5 months (range, 6–12 months). The IgG1 anti-NC16A BP180 autoantibodies decreased to a mean nadir of 3.9 IU (mean 13.6% of T0; range of 1.7–6.4 IU; WSR p value < 0.05 compared to T0) at a mean of 10.2 months (range 6–12 months), while IgG2 anti-NC16A BP180 autoantibodies decreased to a mean nadir of 25.2 IU (mean 23.4% of T0, range 0–86.2 IU; WSR p value < 0.05) at a mean 8.2 months (range 5–12 months). The 2 subjects with detectable anti-NC16A BP180 IgG3 decreased to a mean nadir of 1.5 IU (mean decrease 3.2% of T0) at 3 months. Four subjects were found to have detectable IgG4 antiNC16A autoantibodies that decreased to a mean nadir 4.3% of T0, range 0.2–115 (WSR; p value = 0.063) at 8.3 months however the lowest values did not reach a statistical significance (p = .0625, WRS). No significant difference was seen between the % decrease from T0 in total IgG and the decrease for the other IgG subclasses. Similarly, analysis of the time at which the nadir value occurred revealed, no difference between the time to the nadir for total IgG and the IgG subclass autoantibodies (Fig. 1). There was no difference noted in the change of total IgG or IgG subclass reduction between the 5 subjects with no disease flare and the 2 with a disease flare. The subjects who flared however did have an increase in the level of IgG anti NC16A BP 180 at the time of the flare [10]. Analysis of the levels of IgG anti NC16A domain BP180 specific autoantibodies in subjects with BP for one year after rituximab therapy revealed that treatment did not preferentially decrease any specific IgG subclass of autoantibodies as compared to the total
[1] Giudice GJ, Emery DJ, Zelickson BD, Anhalt GJ, Liu Z, Diaz LA. Bullous pemphigoid and herpes gestationis autoantibodies recognize a common noncollagenous site on the BP180 ectodomain. J Immunol 1993;151:5742–50. [2] Zillikens D, Mascaro JM, Rose PA, Liu Z, Ewing SM, Caux F, et al. A highly sensitive enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid. J Invest Dermatol 1997;109:679–83. [3] Schmidt E, Obe K, Brocker EB, Zillikens D. Serum levels of autoantibodies to BP180 correlate with disease activity in patients with bullous pemphigoid. Arch Dermatol 2000;136:174–8. [4] Hofmann S, Thoma-Uszynski S, Hunziker T, Bernard P, Koebnick C, Stauber A, et al. Severity and phenotype of bullous pemphigoid relate to autoantibody profile against the NH2- and COOH-terminal regions of the BP180 ectodomain. J Invest Dermatol 2002;119:1065–73. [5] Laffitte E, Skaria M, Jaunin F, Tamm K, Saurat JH, Favre B, et al. Autoantibodies to the extracellular and intracellular domain of bullous pemphigoid 180, the putative key autoantigen in bullous pemphigoid, belong predominantly to the IgG1 and IgG4 subclasses. Br J Dermatol 2001;144:760–8. [6] Schmidt E, Seitz CS, Benoit S, Brocker EB, Goebeler M. Rituximab in autoimmune bullous diseases: mixed responses and adverse effects. Br J Dermatol 2007;156:352–6. [7] Joly P, Mouquet H, Roujeau JC, D’Incan M, Gilbert D, Jacquot S, et al. A single cycle of rituximab for the treatment of severe pemphigus. N Engl J Med 2007;357:545–52. [8] Kasperkiewicz M, Shimanovich I, Ludwig RJ, Rose C, Zillikens D, Schmidt E. Rituximab for treatment-refractory pemphigus and pemphigoid: a case series of 17 patients. J Am Acad Dermatol 2011;65:552–8. [9] Khosroshahi A, Carruthers M, Deshpande V, Unizony S, Bloch D, Stone J. Rituximab for the treatment of IgG4-related disease: lessons from 10 consecutive patients. Medicine (Baltimore) 2012;91:57–66. [10] Hall RP, Streilein RD, Hannah D, McNair P, Fairley JA, Ronaghy A, et al. Association of serum B cell activating factor (BAFF) level and proportion of memory and transitional B cells with clinical response after rituximab treatment of bullous pemphigoid patients. J Invest Dermatol 2013. http:// dx.doi.org/10.1038/jid.2013.236 [advanced online publication 27 June 2013].
Arash Ronaghy, Robert D. Streilein, Russell P. HallIII* Department of Dermatology, Duke University Medical Center, Durham, NC 27710, USA *Corresponding author at: Box 3135, Duke University Medical Center, Durham, NC 27710, USA. Tel.: +1 919 684 3110; fax: +1 919 684 3002 E-mail addresses: [email protected] [email protected] (R.P. Hall III). Received 3 July 2013 Accepted 28 November 2013