B-Cell Depletion in Immune Thrombocytopenia

Bertrand Godeau

B cells play an important role in the immune response and can lead to the development of autoimmune diseases and particularly immune thrombocytopenia (ITP). A rational approach to ITP treatment could involve B-cell depletion such as with rituximab. Rituximab is a chimeric monoclonal antibody directed against CD20 molecule. It has direct effects on antibody production and indirect effects on cellular immunity. Rituximab demonstrated an overall response rate of 62.5% that lasted from 2–48 months. The ability of rituximab as an effective splenectomy-avoiding option was recently confirmed in a meta-analysis of randomized clinical trials and observational studies including 368 patients with an overall response rate of 57%. However, the estimated 5- year response is only 21% in adults. Rituximab appears to be well tolerated, but we lack studies of long-term tolerance. The optimal time to administer rituximab for ITP remains unclear. There is consensus to administer corticosteroids or intravenous immunoglobulin (IVIg) as first-line therapy in ITP. A panel of experts was unable to formulate a clear strategy for the respective place of splenectomy, thrombopoietin-receptor agonists, and rituximab as second-line treatment. Among new-generation CD20-targeted therapy, only veltuzumab has been tested for ITP. Preliminary study suggests that it could have similar efficacy to rituximab. Options other than anti-CD20 treatment may modulate and/or inhibit the B-cell compartment. Several monoclonal antibodies (MoAbs) directed against different B-lymphocyte receptors or structures implicated in the cooperation between B and T lymphocytes have been successfully tested in various autoimmune diseases. Testing these options in ITP will be an exciting challenge. Semin Hematol 50:S75–S82. C 2013 Published by Elsevier Inc.

mmune thrombocytopenia (ITP) is a rare disease involvement of CD8 T-cell destruction of platelets characterized by low platelet count resulting in as well as breakdown of tolerance perhaps because I autoimmune phenomena. For a long time the of regulatory T-cell deficiency.2 disease was considered mainly related to autoanti- B cells play an important role in the immune bodies against platelet glycoproteins (GPs). There- response. Their major role is to produce antibodies.3 fore, the pathophysiologic aspects are complex and However, B cells are also efficient antigen-presenting probably reflect the heterogeneity of the disease.1 cells for T cells. This role appears important for Impaired platelet production has been long sus- presenting low concentrations of antigens. The pected and recently confirmed by a strong response process involves molecules such as B7/CD28 and obtained with use of thrombopoietin receptor ago- CD40/CD40 ligands on the surface of B cells and T nists. In addition, in vitro studies suggested the cells, respectively, which helps expand the number of CD4þ T cells. B cells secrete cytokines such as interleukin-1 (IL-1), IL-10, IL-6, interferon-g, and Centre de re´fe´rence des cytope´nies autoimmunes de l’adulte, CHU Henri Mondor, Assistance Publique-Hopitauxˆ de Paris, Cre´teil, tumor necrosis factor-a (TNF-a), which activate France. macrophages, dendritic cells, and immunoregulatory Publication of this article was supported by the International Coop- cells. These various functions explain why disor- erative ITP Study Group (ICIS). Conflicts of interest: Bertrand Godeau receives research support from dered B-cell function can lead to the development of Roche France; he participates in advisory boards or received autoimmune diseases, by the production of patho- consultant fees from AMGEN, GSK, SuppreMol, LFB. genic autoantibodies and a larger dysregulation of Address correspondence to Bertrand Godeau, MD, Centre de re´fe´rence des cytope´nies autoimmunes de l’adulte, CHU Henri Mondor, the immune response. Finally, B cells play an Assistance Publique-Hopitauxˆ de Paris, UPEC, 51 avenue du important role in normal body architecture and the Mare´chal de Lattre de Tassigny, 94000 Cre´teil, France. E-mail: development of secondary lymphoid organs. [email protected] 0037-1963/$ - see front matter The role of B cells in the ITP pathology was first & 2013 Published by Elsevier Inc. reported more than 50 years ago with the Harrington– http://dx.doi.org/10.1053/j.seminhematol.2013.03.014 Hollingsworth experiment demonstrating that platelet

Seminars in Hematology, Vol 50, No 1, Suppl 1, January 2013, pp S75–S82 S75 S76 B. Godeau destruction was related to a plasma-derived factor.4 cases. However, antibody titers are reduced with The factor was later identified to be antiplatelet anti- anti-pneumococcal vaccination.11 bodies directed against GPs, mainly GPIIb/IIIa and The mechanisms of action of rituximab in ITP are GPIb/IX. However, only about 50% of patients show not fully understood and are complex. Rituximab has platelet antibodies. Failure to detect antibodies might direct effects on antibody production but indirect reflect poor test sensitivity but could be related to effects on cellular immunity. mechanisms of platelet loss not involving B cells. The titers of platelet autoantibodies can decrease Thus, a rational approach to ITP treatment could markedly or even became undetectable after B-cell involve B-cell depletion such as with rituximab. depletion. Titers of platelet-associated antiplatelet antibodies may differ between responders and non- responders to treatment, and the timing of decreased RITUXIMAB FOR ITP titers of platelet-associated antiplatelet antibodies may be associated with increased platelet count.12 Human CD20 However, the autoantibody response is not constant Human CD20 is a 33- to 37-kd non-glycosylated in responders, which suggests that other mecha- phosphoprotein exclusively expressed in the B-cell nisms may be involved. Studies by Stasi and col- hematopoietic lineage.5 Its exact function and natu- leagues13,14 showed that the rituximab response is ral ligands are unknown. CD20 is closely associated related to changes in the T-cell compartment, in with the B-cell antigen receptor. It is not expressed particular a resolution of skewed distribution of in hematopoietic stem cells or plasma cells, so human T-cell receptors, changes in T-helper subsets depleting a B-cell lineage with a monoclonal anti- and improved regulatory T-cell function. In non- body (MoAb) directed against CD20 will not affect responders, CD8 T-cell–mediated platelet or mega- the development of new B cells and is not associated caryocyte destruction could be involved. In some with a significant decrease in immunoglobulin (Ig) patients, both antibody and cellular elements may production against pathogens. In theory, this sce- contribute to thrombocytopenia.15 nario limits the risk of infectious complications and A study of the effect of rituximab on the spleen hypogammaglobulinemia. implied the involvement of other mechanisms. The spleen plays an important role in the pathophysio- logic aspects of ITP. It is an important site of platelet Development of Rituximab and Mode(s) antibody production and of platelet destruction, as of Action shown by isotopic studies and the efficacy of Rituximab is a human-to-mouse chimeric anti-CD20 splenectomy with high response in ITP. Audia MoAb initially developed 20 years ago to treat lym- et al16 showed that in some patients with no phoma. The mode of depletion of CD20þ cells is not response to rituximab who later underwent splenec- completely understood, but in vitro and animal tomy, rituximab had eliminated nearly all circulating studies have demonstrated that antibody-dependent and splenic B cells but was not sufficient for a cellular cytotoxycity (ADCC) is crucial.6 Complement- clinical response. The authors showed plasma cells dependent cytotoxicity (CDC) and apoptosis via a persisting in the spleen. Moreover, patients with no direct effect of MoAb binding to CD20 may be also response to rituximab therapy show a marked involved. ADCC is mediated by effector cells express- imbalance in ratio of T-helper cells type 1 to regu- ing Fc-g receptors, such as natural killer cells and latory T cells. Mahe´vas et al17 analyzed the splenic B- macrophages. The degree of ADCC depends on the cell compartment in ITP patients without response effector target cell ratio, and polymorphism of the to rituximab. The authors confirmed that plasma Fc-g receptor on effector cells could determine the cells were the major residual splenic population and efficacy of rituximab. a fraction were autoreactive, which accounted for Rituximab was also developed for use with vari- persistent disease and failure of rituximab treatment. ous autoimmune diseases such as rheumatoid arthri- Using gene profiling and microarray assay, the tis (RA), systemic lupus erythematosus (SLE), and authors demonstrated a unique long-lived plasma autoimmune cytopenia.7–9 It induces continual, cell phenotype for some plasma cells. rapid, profound and prolonged B-cell depletion. However, the B-cell number returns to normal after 6–12 months, with repopulation of the B-cell pool Efficacy of Rituximab in ITP characterized by the appearance of immature B cells; More than 10 years ago, Stasi et al18 reported the the number of CD27þ memory B cells remains results of the first open prospective study of 25 reduced for up to 2 years.10 The long-lived plasma adults with chronic ITP who received 4 weekly cell compartment does not appear to be affected by infusions of 375 mg/m2 rituximab. The authors rituximab, and IgG level remains stable in most found an overall response rate of 52%. Six years B-cell depletion in ITP S77 later, Arnold et al19 conducted an extensive review ITP. The median time to response was 35 days of the literature, involving 313 patients. The overall (range, 7–112 days) and was similar to that with response rate was 62.5% and lasted from 2–48 the standard regimen. The 12- and 24-month cumu- months. Toxic effects were found; 2.9% of patients lative relapse-free survival rates were 61% and 45%, died, but some of the deaths could have been due to respectively. This study suggested that low-dose ITP rather than the treatment itself. From these rituximab is active but has moderate long-term encouraging results, an open prospective study effect. A retrospective study recently conducted in was conducted to assess the efficacy and safety of France included 107 patients with chronic ITP and rituximab in 60 non-splenectomized adults who compared the standard dose and the ‘‘RA’’ regimen were candidates for splenectomy.20 Two years after of two fixed doses of 1000 mg on days 1 and 15.29 rituximab therapy, 40% of patients showed sustained This schema was validated in RA in prospective platelet count of 30,000/L or more. The ability of controlled study and is also used for other auto- rituximab as an effective splenectomy-avoiding immune disease such as SLE or anti-neutrophil option was recently confirmed in a meta-analysis of cytoplasmic antibody-related necrotizing vasculi- randomized clinical trials and observational stud- tis.7,30 For ITP, overall response rate and tolerance ies.21 The overall response rate was 57% for 368 were similar with both regimens, so this schema may non-splenectomized patients after rituximab ther- replace the standard regimen as a more convenient apy. On univariate or multivariate mixed-effects schedule for the patient. meta-regression, age was the most relevant associ- ated factor, with better results in young patients. The use of rituximab as first-line therapy in patients Repeated Courses with newly diagnosed ITP was investigated in a Because relapse is frequently observed after the prospective study comparing dexamethasone alone initial response, Hasan et al31 tested the value of re- and dexamethasone plus 4 weekly injections of treatment with rituximab with the same schedule as rituximab at a ‘‘standard dose’’ of 375 mg/m2.22 the initial course or a doubling dose with and The addition of rituximab to dexamethasone signifi- without cyclophosphamide. Unfortunately, neither cantly improved the long-term efficacy rate, with a combining rituximab with cyclophosphamide nor sustained response of 85% at 6 months as compared doubling the rituximab dose increased the response with 39% with dexamethasone alone. At a median rate, and in 75% of patients, re-treatment induced follow-up of 20 months, approximately 75% of the similar response. Because the safety of repeated initial responders showed a lasting response. Unlike rituximab infusions is unknown, in our opinion, this these good results, an anti-CD20 strategy appears not therapeutic strategy should be avoided and reserved to be indicated as standard first-line therapy in most for select patients with disease refractory to other guidelines, and corticosteroids alone remain the gold therapies. standard.23,24 Only few data on the long-term response of rituximab are available because the duration of Rituximab With Other Treatments follow-up has been limited in most reported studies. As indicated previously, a controlled randomized A retrospective study of 72 adults with response of study demonstrated that dexamethasone alone was at least 1 year and 65 children with response of any less effective than dexamethasone with rituximab duration showed an initial overall response rate of for ITP.22 Unfortunately, results of a randomized 57% in adults and children and 5-year estimates of study comparing rituximab alone and rituximab plus persistent response of 21% and 26%, respectively.25 dexamethasone have never been reported. However, Initial complete response and prolonged B-cell in a single-center pilot study, 36 adults received depletion were significantly associated with sus- 4 weekly infusions of 375 mg/m2 and three 4-day tained response. cycles of high-dose dexamethasone at 2-week inter- vals32; the overall response rate was 86%. After a median follow-up of 14 months, 74% of responding The Optimal Therapeutic Schedule of patients showed a continual response. Five patients Rituximab for ITP showed continual complete responses from 24 to 50 months. From the history of patients previously Dose(s) treated in the same unit with the standard regimen Most studies used the ‘‘standard’’ dose used to (ie, 4 weekly injections of 375 mg/m2), rituximab treat lymphoma (ie, 375 mg/m2); however, some plus dexamethasone appeared to be active and authors proposed a fixed dose of 4 weekly injections superior to rituximab alone. The response was better of 100 mg.26–28 The most recent study with this dose for patients with a shorter duration of ITP (o1–2 prospectively included 48 adults with symptomatic years). Prospective studies should be conducted to S78 B. Godeau confirm these promising results and to better assess been observed mainly in patients receiving the the tolerance of this therapeutic schedule. treatment for chronic lymphocytic leukemia. This risk for patients receiving rituximab for ITP is rare. Safety of Rituximab in ITP Neutropenia is usually transient and is not compli- cated by severe infection. Its mechanism is not well We need better knowledge of the safety of understood.38 rituximab in the short and long term. From the literature, rituximab appears to be well tolerated, Reduced Ig Levels but we lack studies of long-term tolerance. A large prospective registry of patients receiving treatment In theory, this risk should be rare because CD20 is in daily practice for RA was established in France.33 not expressed on mature B cells. However, we The registry included 1,303 patients who were occasionally observed patients with this complica- 25 monitored for a mean of 1.2 Ϯ 0.8 years. The rate tion a long time after rituximab administration. of severe infections was 5 per 100 patient-years and Ensuring that this complication is not the first appeared similar to that for patients with RA receiv- manifestation of common variable immunodefi- ing other biotherapy. However, these reassuring data ciency (CVID) syndrome is difficult because auto- cannot be extrapolated to ITP. Moreover, in real life, immune cytopenia is a frequent complication of 39 the side effects of rituximab, particularly after a long CVID. Even if the incidence of this complication delay, are probably underreported. To answer this may be low, regular monitoring of IgG levels and for important question, a prospective registry of ITP a long time may be useful. patients receiving rituximab was established in France 2 years ago.34 Only patients previously naive Suboptimal Vaccine Response for this treatment were included. Currently, 250 Some studies suggest impaired humoral immune patients have been included. A follow-up of 5 years response to vaccines with rituximab treatment.11 is planned. This registry should allow for a better Therefore, logically, vaccines should be administered picture of tolerance of rituximab in ITP. at least 2 to 4 weeks before the first rituximab Several risks of rituximab treatment are well infusion in non-immunized patients. Anti-pneumo- 35 known and some can be easily prevented. coccal, -haemophilus, and -meningococcus-C vacci- nations appear crucial for non-splenectomized Infusion-Related Reactions patients. If a splenectomy is required in the year Rare fatal reactions have been reported in patients after rituximab infusions, a suboptimal vaccine with RA, and physicians should not forget that response can be prolonged for 1 year. premedication with 100 mg methylprednisolone should be systematically administered, according to Optimal Time to Administer Rituximab for ITP health authorities recommendations. The consensus is to administer corticosteroids or intravenous Ig (IVIg) as first-line therapy in ITP.23,24 Infections and Virus Reactivation However, the effect of these treatments is frequently only transient, and most patients show relapse. The This is a concern. This risk is difficult to quantify, best second-line treatement is still debated and not even if the data for patients receiving rituximab for consensual. A panel of experts was unable to RA are reassuring (see above). Rituximab can be formulate a clear strategy for the respective place associated with virus reactivation. This risk appears of splenectomy, thrombopoietin-receptor (TPO-r) to be high for patients with hepatitis B virus (HBV) agonists and rituximab as second-line treatment.23 infection, and cases of fatal fulminant hepatitis have American Society Hematology (ASH) guidelines pro- been reported. Special care should be paid to screen- posed splenectomy (grade 1B) for patients with no ing for HBV. With the presence of markers of HBV, response to corticosteroids24; TPO-r agonists should other therapeutic options should be considered, even be reserved for patients with relapse after splenec- if pre-emptive antiviral treatment could be an option. tomy or those with contraindications to splenectomy An important question is the risk of fatal progressive and for whom at least one other therapy failed multifocal leukoencephalopathy caused by the John (grade 1B). Rituximab may be considered for Cunningham polyomavirus. This serious complica- patients with no response to one line of therapy tion is, fortunately, exceptional and to our knowl- such as corticosteroids, IVIg, or splenectomy (2C). edge, only two cases have been reported.36,37 However, international consensus and ASH guide- lines emphazise the general rule that treatment Neutropenia should always be tailored to the individual patient Late-onset neutropenia occurring usually several and that a recommendation should not replace the months after the administration of rituximab has best physician judgment and patient preference. B-cell depletion in ITP S79

According to these recommendations, some authors NEW ANTI-CD20 ANTIBODY recently proposed a personalized strategy including several variables such as age of the patient, duration Rituximab has greatly improved the outcome of of ITP, presence of comorbidities, results of isotopic patients with B-cell malignancies. However, despite its studies, patient choice, and possible restrictions on success, some patients show no response, which 44 the use of TPO-r agonists and/or rituximab by health points to new-generation CD20-targeted therapy. funding authorities.40 According to this schedule, a Second-generation MoAbs have been ‘‘humanized’’ candidate for rituximab as second-line treatment or developed with a completely human IgG1 tail. could be a patient who refuses splenectomy but Third-generation anti-CD20 MoAbs have, in addition to who prefers treatment for cure, with high risk of a humanized IgG, an ajusted Fc region modified to arterial or vein thrombosis, or with anticipated poor outperform rituximab and to induce different effector compliance. functions. Some third-generation anti-CD20 MoAbs are potent CDC inducers. However, others do not induce CDC but, rather, strongly promote cell death by Rituximab as an Option for Patients With ITP Secondary to apoptosis. These new CD20-targeted MoAbs are Other Diseases mainly developed for B-cell malignancies. Whether they have better activity than rituximab for treating Secondary to Hepatitis C Virus (HCV) Infection. To autoimmune disease remains to be determined. our knowledge, no case of HCV-infection– Among new-generation CD20-targeted therapy, only associated ITP has been treated with rituximab. veltuzumab has been tested for ITP.45 Veltuzumab is a Even if rituximab use is associated with a risk of second-generation anti-CD20 MoAb. It is humanized viral reactivation, this risk is probably low, and and delivered by subcutaneous injection. Compared to rituximab has been used without problems in rituximab, it has a slower off-rate. A multicenter, phase other extra-hepatic manifestations related to HCV I/II study evaluated various doses of veltuzumab in 2 to infection such as mixed cryoglobulinemia or 4 weekly subcutaneous injections as monotherapy for 41 necrotizing vasculitis. ITP patients. Most patients had been intensively treated for chronic disease. Of the 40 patients with Secondary to HIV Infection. As with HCV infection, evaluable data, the overall response rate was 68%. The data are lacking to adequately evaluate the use of median time to relapse was correlated with the initial rituximab for these cases. However, rituximab response and was 14.2 months for patients with an should be avoided in patients with HIV infection initial complete response. The response was 35% at and human herpes virus 8–related Kaposi sarcoma more than 1 year, and two patients showed response because worse disease evolution has been at more than 3 years. Subcutaneous veltuzumab reported. appears to be promising. Further studies combining veltuzumab with other agents such as dexamethasone Secondary to SLE. Several uncontrolled studies of a or as a maintenance regimen could be useful. small number of patients suggested that rituximab could be useful in this field. Previous data suggested that rituximab could be associated with OTHER OPTIONS TO INDUCE B-CELL risk of progressive multifocal leukoencephalopathy DEPLETION in SLE. The French prospective registry reporting Options other than anti-CD20 treatment may mod- the tolerance of rituximab in 136 SLE patients gave ulate and/or inhibit the B-cell compartment. Several 42 reassuring data. MoAbs directed against different B-lymphocyte recep- tors or structures implicated in the cooperation Secondary to CVID Syndrome. The efficacy of between B and T lymphocytes have been tested in rituximab was retrospectively assessed in 22 various autoimmune diseases, particularly RA and SLE. patients with CVID syndrome-associated ITP.43 Testing these molecules in ITP would be attractive. The overall response rate was 85%. Relapse was Belimumab is a human monoclonal antibody that observed in one third of patients after a mean inhibits B-cell activating factor (BAFF), also known as follow-up of 39 months. Re-treatment was B-lymphocyte stimulator (BLyS). BAFF enhances B- effective in most. Severe infections occurred in cell survival; overexpression of BAFF results in one quarter of patients. Because of lack of a severe autoimmune disorders in mice, and patients control group, ensuring that these infections with various autoimmune diseases show elevated were related to rituximab or to the CVID itself is levels of BAFF. Patients with ITP have shown this difficult. However, IVIg replacement appears increased BAFF phenotype, which suggests that new cautious, even in the absence of previous therapeutic options such as belimumab targeting infectious complications. BAFF may be developed for ITP.46 S80 B. Godeau

CD22 is a 140-kd member of the cell-surface which are effective and well tolerated and have a proteins that is expressed by most mature B-cell clear immediate overall response rate as compared lineages. As a co-receptor of the B-cell antigen with rituximab, rituximab is able to reverse the receptor, it contributes to sensitive control of the autoimmune process for disease cure. Identifying B-cell response to antigens. Modulating CD22 activ- patients who are more likely to show treatment ity has been suggested as a therapeutic approach to response is a major goal for the near future. Even if such diseases. For example, epratuzumab is a novel rituximab appears to be well tolerated, better knowl- fully human CD22-targeting monoclonal antibody edge of long-term safety is required. As well, we that induces B-cell apoptosis. It is currently under must determine the best therapeutic schedule and investigation as treatment for connective tissue dis- the potential interest of association with other orders, particularly SLE, with preliminary encourag- therapy such as dexamethasone. Testing the efficacy ing results.8 and tolerance of other drugs modifying B-cell activity T-cell activation depends on close contacts with will be also an exciting challenge. antigen-presenting cells. It requires several signals, particularly an interaction between the B7 family members B7-1 (CD80) and B7-2 (CD86) on the REFERENCES surface of such cells and their ligand CD28, 1. Stasi R. Pathophysiology and therapeutic options in expressed on T cells. CTLA-4 is a second receptor primary immune thrombocytopenia. Blood Transfus. 2011;9:262–73. for B7-1 and B7-2 that is expressed on the membrane 2. Chong BH. ITP: Tregs come to the rescue. 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