The paper by Seitz-Polski et al. (1) in this issue of CJASN addresses the influence of epitope spreading and drug dose on the efficacy of rituximab in patients with primary membranous nephropathy associated with antiphospholipase A2 receptor (anti-PLA2R) antibodies.
Epitope spreading is an immunologic phenomenon whereby an antibody or cellular response to a given antigen may extend from one location on the antigen (epitope) to involve other region(s) of the same antigen (intramolecular spreading) or adjacent antigens (intermolecular spreading) as the immune response matures (2). Experimental evidence that epitope spreading might occur and affect the severity of membranous nephropathy was clearly documented in the Heymann nephritis model of membranous nephropathy in rats (3). In that study, Shah et al. (3) immunized rats with a 236-residue N-terminal fragment of the target antigen megalin to induce antimegalin autoantibodies and severe membranous nephropathy. Sera obtained 4 weeks after immunization showed reactivity to the N-terminal epitope alone, whereas sera obtained at later time points showed intramolecular spreading to other regions of megalin coincident with worsening proteinuria. In the context of primary membranous nephropathy associated with anti-PLA2R antibodies, a substantial proportion of cases was found to exhibit spreading from an immunodominant epitope in the N-terminal cysteine-rich (also known as ricin) domain in all anti-PLA2R–positive cases (4,5) to involve epitopes in the CTLD1 and/or CTLD7 C-type lectin domains of PLA2R (5) (Figure 1). There is also evidence for intermolecular spreading in membranous nephropathy as antibodies to various cellular antigens appear over time (6). Although not unexpected on the basis of the experimental studies cited above and other autoimmune diseases (2), it is important to know if such spreading has an impact on clinical outcome and response to treatment.
In general, epitope spreading produces a more robust immune response to a given antigen. Although this is desirable to fight microbial invasion and cancer spread, it may make an autoimmune condition more resistant to the restoration of self-tolerance and spontaneous remission or drug-induced immune suppression. Such seems to be the case in PLA2R-associated membranous nephropathy. The clinical significance of epitope spreading in membranous nephropathy anti-PLA2R–associated membranous nephropathy was first suggested in a prior cross-sectional study by these authors, in which it was found that those patients that did not spread beyond the cysteine-rich domain (nonspreaders) were younger, had milder disease, were more likely to enter spontaneous remission (45% versus 0.05% in spreaders), and were less likely to require kidney replacement therapy than those that had spreading to CTLD1 and/or CTLD7 (5). The effect of epitope spreading was also examined in a post hoc analysis of the prospective GEMRITUX study, in which PLA2R-associated patients were randomized to receive rituximab in two infusions of 375 mg/m2 at a 1-week interval added to antiproteinuric therapy and compared with patients who received antiproteinuric therapy alone (7). In the GEMRITUX study, there was no significant difference in the primary combined end point of complete or partial remission of proteinuria at 6 months between the rituximab treatment and control groups (8). However, anti-PLA2R depletion occurred in significantly more of the patients treated with rituximab than controls, and significantly more patients treated with rituximab entered remission with extended follow-up (8). As in the 2016 study by Seitz-Polski et al. (5), epitope spreading at baseline as well high anti-PLA2R titers were independently associated with worse outcome in the GEMRITUX study (7). Is this an insoluble problem resulting from a more vigorous immune response, or is there a solution?
The paper in this issue of CJASN by Seitz-Polski et al. (1) attempted to address this problem by conducting another post hoc analysis in which they paired patients from the GEMRITUX study with a series of patients treated in Nice, France who had received a higher dose of rituximab—two infusions of 1 g at a 2-week interval (NICE protocol) (1). Overall, remission occurred in more patients and more rapidly with the higher dose (NICE) protocol; circulating rituximab levels were higher, and CD19 (B cell) counts were lower. As in the previous study, all patients from both cohorts in whom the autoantibodies were directed only at the immunodominant epitope in the N-terminal cysteine-rich domain responded to rituximab regardless of the dose. In contrast, this report demonstrated that, in those patients with spreading to epitopes in the CTL domains, higher doses of rituximab, as administered in the NICE protocol, were necessary to eliminate the autoantibodies and induce remission.
One could argue that those patients with epitope spreading had more severe disease with more proteinuria and therefore, might have lost more of the drug—an mAb—in the urine, and this would have necessitated a higher dose to achieve the therapeutic effect. However, this argument does not hold up, because serum rituximab levels were not significantly different at 3 months in those with and without epitope spreading. Rather, it would seem that sustained levels of the drug are necessary to suppress the B cell clones.
Why not give the higher dose to everyone? After all, side effects, including first-dose and mucocutaneous reactions, hepatitis B reactivation, and progressive multifocal leukoencephalopathy, do not seem to be dose related, and hypogammaglobulinemia has been reported mostly in children with nephrotic syndrome. In addition, the original membranous nephropathy protocol of four doses of 375 mg/m2 at 1- to 2-week intervals was arbitrarily borrowed from lymphoma protocols, and the Mayo studies showed similar outcomes with that protocol and one similar to the NICE patient series (9). However, cost remains a major impediment, and the Bergamo B cell–driven protocol showed that a single dose may be sufficient in some patients and can be repeated as necessary to deplete B cells, suppress anti-PLA2R levels, and induce remission (10). Therefore, judicious use is warranted, and selection of patients for high-dose rituximab needs to be rationalized.
Therefore, does this report from Seitz-Polski et al. (1) help guide use of rituximab in the treatment of membranous nephropathy? Because the major determinants of remission are drug dose and epitope restriction versus spreading at baseline and because measures of anti-PLA2R epitope spreading are not currently available, one wonders if anti-PLA2R titer at baseline could serve as a surrogate (albeit imperfect) marker for spreading. This view is supported by supplemental figures 2 and 4 in the paper by Seitz-Polski et al. (1), which show that spreaders tend to have higher titers and that patients with high titers at baseline do better with the NICE protocol. Therefore, until such time as measures of anti-PLA2R epitope spreading are routinely available, it seems appropriate that those patients with high titers and severe nephrotic syndrome should receive rituximab at doses equivalent to the NICE protocol. This raises yet another unresolved question about anti-PLA2R levels. Although recent studies have begun to refine the cutoff for a positive anti-PLA2R using currently available assays (11), there are no formal studies that distinguish high from low positive values. Seitz-Polski et al. (1) define an ELISA cutoff of 70 RU/ml to distinguish the lower tertile from the upper two tertiles of anti-PLA2R (using the Euroimmun ELISA), and they show in their supplemental figure 4 that 95% of patients with titers >321 RU/ml are spreaders. However, additional studies are needed with sensitivity analyses and a standardized assay to determine if this is an appropriate level.
Does reversal of spreading occur, and if so, what does it signify? Seitz-Polski et al. (1) note that the percentage of epitope spreaders tended to be lower at month 6 in those treated with the NICE protocol. This is an interesting finding that suggests that high-dose rituximab may reverse spreading, and it merits additional study to confirm the finding and determine if it improves outcomes compared with those in patients who fail to experience spreading reversal.
In conclusion, as rituximab and perhaps, next generation B cell–depleting agents rise to become mainstream therapy for severe membranous nephropathy, it is important that we refine our protocols to provide maximum benefit to those patients who need the treatment while limiting cost, overuse, and toxicity. The paper by Seitz-Polski et al. (1) moves the field in that direction by helping to define those patients most likely to need and benefit from high doses. It also highlights the influence of epitope spreading on resistance to treatment and raises expectations that assays to identify patients with spreading may become available in the clinical realm. What it does not do is tell us is if and when redosing is needed and whether those patients with epitope spreading and high anti-PLA2R titers would have done better with retreatment.
Dr. Salant discloses the following: coinventor on the patent “Diagnostics for membranous nephropathy”; royalties from UpToDate, diagnostics for membranous nephropathy; grant support from Pfizer CTI; and consulting fees from Chugai and Sanofi/Genzyme. Dr. Salant is on the data and safety monitoring committee of Pfizer.
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