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M. Mahevas et al.
Introduction
Primary immune thrombocytopenia (ITP) is a bleeding disorder mainly mediated by autoreactive B cells and plasma cells (PC) secreting pathogenic anti-platelet auto- antibodies, eventually leading to accelerated platelet destruction and impaired megakaryopoiesis.1,2 First-line treatments include steroids and intravenous immunoglobulins (IVIg). Because less than 40% of newly diagnosed ITP adults will achieve a spontaneous remis- sion within 12 months after disease onset, second-line treatments are frequently needed.3 Over the past 20 years, the anti-CD20 monoclonal antibody rituximab (RTX) has been considered an off-label second-line option in many countries and most guidelines. RTX leads to an overall response rate of 40% at 1 year.4,5 Whereas an almost complete B-cell depletion is achieved in peripheral blood and in secondary lymphoid organs after RTX in ITP,6 approximately half of the patients do not respond to RTX, which raises many questions and has led to some investigations in the past years.
In ITP, pathogenic antibody-secreting PC are constantly generated in the spleen, mainly through the germinal cen- ter pathway.6,7 Because most of these splenic PC are short- lived and have lost CD20 expression, the clinical improve- ment observed after RTX is thought to result mainly from germinal center depletion, thus limiting PC generation.8,9 However, analysis of spleen samples from ITP patients with failure of RTX revealed that despite complete periph- eral B-cell depletion, residual splenic PC secreting anti- platelet antibody persisted.6 More surprisingly, transcrip- tomic analysis showed that these splenic PC had acquired a long-lived program, similar to bona fide bone-marrow long-lived PC. Quantitatively, the data suggested that B-cell depletion had induced the differentiation of short- lived PC into long-lived ones, rather than the selection of pre-existing long-lived PC, thus providing clues for explaining RTX failure in the context of ITP.6
By using a fate mapping mouse model, we recently demonstrated that B-cell activating factor (BAFF) played a major role in the emergence of these splenic long-lived PC.10 BAFF is a pro-survival key cytokine for the B-cell lin- eage,11 and elevated levels of unconsumed BAFF are observed in serum and spleen after RTX therapy in ITP patients.6 Combining anti-CD20 with four infusions of anti-BAFF antibodies in this mouse model significantly reduced the number of splenic PC, with little impact on bone marrow PC.10
Hence, we hypothesized that combining two fixed doses of 1,000 mg of RTX with five sequential injections of belimumab (Benlysta®, 10 mg/kg dose) could increase the rate of response at 1 year in patients with persistent or chronic ITP by preventing the emergence of autoreac- tive splenic long-lived PC. Here, we report the efficacy and safety of this new strategy in ITP during a prospec- tive phase IIb pilot trial.
Methods
Study design and study drugs
The study was a single-center, single-arm, prospective phase IIb trial (RITUX-PLUS, clinicaltrials gov. Identifier: NCT03154385) investigating the safety and efficacy of RTX at a
fixed dose of 1,000 mg, 2 weeks apart, combined with five intra- venous infusions of belimumab (Benlysta®, 10 mg/kg) at week 0 (W0) + 2 days, W2 + 2 days, W4, W8 and W12 (see Online Supplementary Figure S1). The rationale for the choice of this schedule was based on previous results obtained in a mouse model showing that BAFF inhibition should start early.10 The belimumab dosing was similar to the one approved in systemic lupus erythematosus. Research was conducted in accordance with the Declaration of Helsinki and was approved by the Comité de Protection des Personnes Ile-de-France VI.
Inclusion and exclusion criteria
Inclusion and exclusion criteria are reported the Online Supplementary Appendix.
Primary endpoint
The primary endpoint was the total number of patients achieving an overall response (complete response [CR] + response [R]) at W52. CR was defined by platelet count >100x109/L and R by platelet count 30-100x109/L with at least a 2-fold increase from baseline according to international defini- tions.12 Patients who required any other treatment for ITP including rescue therapy more than 6 weeks after inclusion were considered non-responders regardless of the platelet count.
Secondary endpoints
Secondary endpoints were the number of patients achieving an overall response (CR+R) initially and at W12, W24, W36, number of bleeding events, number of patients showing severe hypogammaglobulinemia (γ-globulin level <4 g/L at W24, W36, W52), duration of severe hypogammaglobulinemia, variation in γ-globulin subclass levels during the study, and number of severe infections requiring hospitalization during the study.
Adverse events
Adverse effects were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) v5.0 .
Immunological analysis, antibody titer tests, free B-lymphocyte stimulator assay
Phenotype of circulating T- and B-cell subpopulations was analyzed by flow cytometry at W0, W4, W12, W24, W36 and W52 for every included patient and in a prospective control cohort of ITP patients not included in this trial who received two infusions of fixed-dose RTX (1,000 mg) at W0 and W2 after pre- medication with 100 mg intravenous methylprednisolone as a standard of care for ITP (see Online Supplementary Methods, Online Supplementary Table S1, and Online Supplementary Figure S2 for gating strategy). Antibody titers for pneumococcal, tetanus, measles vaccine were measured by enzyme-linked immunosorbent assay (ELISA). Serum-free BAFF level was measured using the assay developed by Glaxo Smith Kline phar- maceuticals (see Online Supplementary Appendix).
Direct monoclonal antibody-specific immobilization of platelet antigen assay
Anti-platelet antibodies on patient platelets were detected by using the monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay (ApDia, Turnhout, Belgium).
Statistical analysis
The statistical analysis was performed as described in the Online Supplementary Methods.
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