Simplified regimen of combined low-dose rituximab for autoimmune encephalitis with neuronal surface antibodies

Autoimmune encephalitis (AE) is a new spectrum of immune-mediated disorders in central nervous system (CNS), characterized by pathogenic autoantibodies against neuronal surface or intracellular proteins [1]. Up to now, autoantibodies against neuronal proteins, especial neuronal surface antibodies (NSAbs), have been linked to more than 15 AE subtypes, such as anti-N-methyl-D-aspartate receptor (NMDAR)-AE, anti-leucine-rich glioma-inactivated-1 (LGI1)-AE and anti-contactin-associated protein-like-2 (CASPR2)-AE, making up the majority of seropositive AE subtypes [2]. Among them, most common NMDAR-AE, predominating in young women and children, mainly led to psychiatric symptoms, amnesia, epileptic seizures, reduced levels of consciousness and abnormal movements [3]. LGI1-AE, particularly affecting middle-aged or elderly patients, frequently caused confusion, short-term memory deficits, faciobrachial dystonic seizures and hyponatremia [4]. CASPR2-AE is mostly observed in elderly men, usually presented LGI1-like encephalitis and peripheral nerve hyperexcitability (neuromyotonia and myokymia) [5]. Despite heterogeneity of clinical features mediated by different antibodies in AE, the characterized manifestations were always involved in psychosis, cognitive impairments and seizures, far beyond motor dysfunctions [6]. NSAbs against surface antigens, such as NMDAR, LGI1 and CASPR2, may directly affect the targeted protein and cause clinical disturbances by blocking functions, interfering with synaptic protein interactions, or subsequent alterations of synaptic density. Furthermore, the underlying autoimmune processes may also lead to irreversible structural damages, as well as severe, progressive and refractory symptoms [7].

Treatment options for AE with NSAbs, ranging from broadly immunosuppressive agents to those targeting antibody-mediated pathogenesis, are mainly focused on achieving both better outcomes and fewer relapses. Common first-line immunotherapeutic agents for acute treatment include methylprednisolone pulse therapy (MPPT), intravenous immunoglobulin (IVIG), plasma exchange (PLEX) or combinations with less specificity for pathogenesis, presenting short-term efficacy during administration. The addition of a second-line agent such as rituximab, cyclophosphamide or combinations will be initiated, if there is no meaningful clinical or radiological response to optimized first-line treatment after 2–4 weeks [8]. Then, a steroid-sparing therapy or a bridging strategy of oral prednisone with a gradual taper overlapping with azathioprine or mycophenolate mofetil (MMF) after completing acute therapy, should be implemented for sustained immunosuppression [9]. There are no established guidelines for AE with NSAbs treatment so far, and the traditional protocol is often complicated and empirically performed according to patient status and clinician opinion. Given the balance between efficacy and safety, an aggressive and practicable approach of immunotherapy after definite diagnosis is critical for AE patients [10].

Recently, a retrospective study based on real-world data has indicated that high-dose rituximab (at least 1 g once) is the most frequent second-line immunosuppressive agent used in AE with NSAbs, but fails to present significant privilege in combination with first-line treatment because of more severity at baseline and complicated addition of drugs in the rituximab cohort, according to scores of modified Rankin Scale (mRS) [11]. Moreover, mRS, initially designed to measure motor function for stroke, has limitations for assessing non-motor deficits of AE mainly presenting psychosis, amnesia and seizures, thereby probably misleading objective evaluation of clinical outcomes. Therefore, a new specialized scale, named the Clinical Assessment Scale for Autoimmune Encephalitis (CASE), has been developed to rate the severity of AE comprehensively [12], and validated for AE with NSAbs in Chinese patients, presenting more sensitive to clinical changes than mRS [13, 14]. Meanwhile, up to now, the infusion regimen, optimal dosage and clinical benefit of combined rituximab for AE with NSAbs treatment still need to be elucidated. Here, we performed a retrospective study to assess the clinical outcomes for patients with NSAbs-associated AE, who were treated with common first-line medications only or with first-line medications and low-dose rituximab (an induction with 100 mg rituximab once a week for 3 cycles, followed by reinfusion 100 mg every 6 months at least for 1 year). We used improvement of CASE scores as the primary endpoint, and alterations in mRS, the Mini-mental State Examination (MMSE), the patient and caregiver Neuropsychiatric Inventory (NPI) scores as the secondary endpoints. We also evaluated clinical relapses, glucocorticoid reduction and adverse effects from different treatments. Without special notation, we will use the term “AE” to refer to AE with NSAbs only in the present study.

In present study, the 3 main findings about rituximab treatment for AE are that: (1) our simplified regimen of low-dose rituximab (100 mg once) combined with common first-line therapy significantly accelerates comprehensive short-term recovery within 1 year, as well as markedly contributing to long-term improvement even after at least 1 year. (2) Our refined protocol of rituximab infusions leads to faster oral prednisolone gradual taper and withdrawal, in parallel with markedly sustained clinical remission and reduced relapses. (3) Opportunity of rituximab schedule shows earlier initiation with better improvement, while frequency of first-line therapy has no influence on satisfactory outcome with rituximab combination.

As we know, self-reactive B cells are subject to the processes of negative selection for elimination, such as deletion, receptor editing and induction of anergy, throughout the development in the bone marrow and spleen [24]. However, emerging data suggest defective B cell tolerance checkpoints in several AE (such as NMDAR-AE, LGI1-AE and CASPR2-AE), increasing autoreactive immature B cells that are not removed but can be activated and enter germinal centers [25, 26]. Several mechanisms may contribute to the loss of B cell tolerance in peripheral lymph nodes in the context of tumor ectopic expressions or potential viral infections, particularly by inducing B cell-intrinsic Toll-like receptor (TLR) signal together with B cell receptor (BCR) ligation, or activating T helper (Th) cell with same antigen stimulation, thereby leading to consecutive B cell clonal expansion, class switch, affinity maturation and NSAbs production [24, 27, 28]. Subsequently, activation of innate immune-mediated cytokines and TLR ligands leads to disruption of the blood brain barrier (BBB), allowing infiltration of autoreactive memory B and plasma cell, followed by proliferation with B cell activating factor (BAFF), and releasing large amounts of NSAbs in CNS [29]. Among them, anti-NMDAR antibodies are mainly IgG1 subclass, leading to rapid reduction of neuronal surface NMDAR by cross-linking, internalization and degradation, while antibodies against LGI1 and Caspr2 are predominantly IgG4, inducing neuronal dysfunction by interrupting the trans-synaptic binding of LGI1 or Caspr2 to its postsynaptic receptor a disintegrin and metalloprotease 22 (ADAM22) and likewise ADAM23 at the presynaptic site, thus causing a series of clinical phenotypes [30, 31].

Rituximab exerts therapeutic effect through its target, cluster of differentiation 20 (CD20), an integral membrane protein mainly expressed on B lymphocytes. During the autoimmune pathological process, the expression of CD20 is progressively increased in B cells at different developmental stages and sustainedly expressed at a high level on the surface of antibody-specific memory B cells and plasmablasts [32]. In NSAbs-associated AE, CD20 may act as a physically coupled link to BCR and other surface molecules or cytoplasmic proteins, such as major histocompatibility complex class II (MHCII), CD40 molecules, and tyrosine kinases, thereby regulating cell cycle progression and proliferation of B lymphocytes [33]. Moreover, activated CD20 + B cells can present same specific antigens to T lymphocytes in association with MHC molecules in the presence of various costimulatory factors, thus promoting T cells activation and differentiation. Subsequently, these T cells can produce a variety of cytokines and chemokines to regulate the maturation and migration of peripheral immune effectors, such as Th, CD8 + T and myeloid cells, secreting a range of proinflammatory mediators to induce neuroinflammation within the CNS parenchyma [34]. Therefore, rituximab may inhibit neuroinflammation via targeting CD20 + B lymphocytes, which results in beneficial effects for AE treatment.

Although instances of spontaneous recovery without immunotherapy have also been reported occasionally in AE, the disease is mostly presented as a progressively monophasic process with rare recurrence (approximately 10–20%) but apparent sequelae, suggesting the irreversible neuronal damages, and advocating the necessity for prompt and persistent interruption of pathogenic immune activity [35]. Traditional AE first-line therapy, including corticosteroids, IVIg, and PLEX, has limitation of less specificity for pathogenesis or shorter maintenance for treatment, while oral prednisone for bridging and steroid-sparing, azathioprine or MMF for sustained immunosuppression, both have common deficiency of less specificity, as well as complicated diverse regimen and continuous adverse effects [8, 9]. Proposals derived from recent systematic review for AE treatment are favored in early initiation of immunotherapy and addition of second-line agents, thereby resulting in better functional outcomes and lower relapses with manageable side effects [36]. Given the largely B cell-secreted antibody mediating the disease pathogenesis, it should be considered that a combination of immunotherapeutic agents targeting B cells may be urgently required for a more efficient regimen of AE treatment [37]. Therefore, rituximab is preferentially selected in second-line agents due to substantially special efficacy and relatively reliable safety.

Rituximab as a second-line agent for AE, initially approved for treatment of lymphoma, is a human/murine chimeric monoclonal antibody directed against a differentiation glycoprotein CD20 participating in B lymphocytes activation and proliferation. After binding to CD20 on the B cells surface, rituximab specifically depletes target B lymphocytes (such as naïve B cells, memory B cells and some plasmablasts) by antibody-mediated cellular toxicity, complement activation and induction of apoptosis, thereby reducing B cells response, and causing therapeutic immunosuppression [38]. Currently, activation of self-reactive B cells and their subsequent proliferation and differentiation into auto-antigen reactive memory B cells and autoantibody-secreting plasma cells, play pivotal pathogenic roles in antibody-mediated neurological diseases, such as AE, neuromyelitis optica spectrum disorders (NMOSD) and myasthenia gravis (MG). Therefore, the off-label use of rituximab for deleting the antigen-specific memory B-cell populations and hence preventing the formation of new plasmablasts which secrete the pathogenic antibodies, was gradually emphasized and presented potential advantages in AE treatment [34]. Consensus criteria on the appropriate time to initiate a second-line agent such as rituximab are yet to be established in AE, but a quick procession is favored, regardless of the response to first-line therapy. When rituximab is used since the acute setting, it may have the added benefit of a potentially faster onset of action, and also serve as a bridging therapy to prevent early relapses that might happen if immunosuppression is abruptly discontinued, as well as an optimal alternative for a sustained immunosuppressant [18]. Thus, the previously complex regimen for AE might be simplified and refined as combination of regular rituximab infusions with conventional first-line therapy.

In reference to a variety of researches about rituximab treatment in AE, there is great heterogeneity concerning dosages. Currently, the empirical protocols for AE are mainly derived from other disease processes such as lymphoma or rheumatoid arthritis (RA), including 375 mg/m² weekly for 4 consecutive weeks or two doses of 1000 mg 2 weeks apart, then followed by reinfusions at fixed intervals for immunosuppressive maintenance, because of circulating B cell below the detectable range for 6–8 months after administration [39]. Moreover, the optimal dosage of rituximab for balancing between safety and efficacy are still ambiguous in AE treatment, and empirical off-label attempt primarily comes from high-dose therapy for lymphomas, usually exerting more medical expenses and serious adverse events [40]. Indeed, the dysfunctional B cells commonly present with normal circulating count in autoimmune diseases, which is different from the high tumor burden in lymphomas, and low-dose rituximab seems to be sufficient and effective for complete depletion of peripheral CD20 + B cells [41]. Recently, reduced low-dose of 100 mg rituximab per infusion for treatment has been tried in some neurological autoimmune disorders, such as NMOSD, MG, multiple sclerosis (MS) and neuro-Behçet’s disease (NBD), with the protocol of induction per week for 3 cycles, then followed by reinfusions at regular intervals. The approach still presented good responsive in depleting B cells, improving clinical symptoms and preventing relapses with favorable side-effect and medical cost [16, 42‐44]. Thus, in present study, we performed a simplified regimen in AE treatment, including regular induction of 3 cycles for acute and bridging management, and subsequent reinfusions (100 mg once) at fixed interval (every 6 months) for sustained immunosuppression.

Meanwhile, the scales for evaluating clinical severity and therapeutic response in AE were also continuously improving and updating. Currently, because of no special tools for AE assessment, the modified Rankin scale (mRS) was widely applied to measure neurological severity and outcome [18, 20]. As we know, the mRS was primarily developed and mainly weighted for estimating prognosis of motor function in acute stroke management, while patients with AE usually presented a variety of symptoms beyond motor deficits, including behavioral changes, memory impairments, seizures, speech disorders, abnormal movements, decreased consciousness, and cerebellar ataxia, which might also interfere with each other in evaluation [12, 45]. Some other scales for special functional domains, such as MMSE for cognitive damages and NPI for neuropsychiatric symptoms, still had obvious limitations of lack diversity in assessment for AE ^{[22. 23]}. Recently, prognostication and estimation tools specifically developed for AE, such as CASE scale with description in detail and validation in practice, might help to select those patients required for more aggressive immunotherapy, and comprehensively and accurately evaluate their clinical outcomes [12]. Hence, according to severity designed previously as mRS scores ≥ 3 or CASE scores ≥ 5, we retrospectively chose moderate and severe AE patients received first-line therapy with or without combination of low-dose rituximab in this study, and also discussed changes of CASE, mRS, MMSE, patient and caregiver NPI scale as well as glucocorticoid dosage and relapses between different cohorts.

As we found during the follow-up, compared to control cohort with common first-line therapy for AE, combined low-dose rituximab cohort not only showed much better outcome in CASE scale evaluation even 1 year later, but also significantly accelerated improvement in CASE, mRS, MMSE, patient and caregiver NPI from baseline within 1 year, as well as markedly reducing occurrence of relapse and oral prednisone dosage, indicating the potential privilege of our simplified regimen of low-dose rituximab in both long-term and short-term prognosis, along with sustained immunosuppression. Meanwhile, longitudinal self-control analysis in both groups also revealed continuously marked amelioration in a series of scales from baseline during at least 1 year, whereas the persistent improvement might be presented even more than 1 year in combination of rituximab. Moreover, further analysis in rituximab cohort showed no difference in any clinical outcomes between combination with single first-line and with repeated first-line treatment (≥ 2 times), while compared with delayed combination with rituximab subgroup (> 3 months), early initiation of combination (≤ 3 months) might achieve better improvements in CASE and MMSE assessments.

This study was limited by its uncontrolled design without comparison with natural course of AE or other dosing regimens, as well as retrospective observational analysis, small sample size and limited follow-up time, and a bias in selecting patients could not be completely ruled out. The data were collected during routine clinical practice rather than a formal study setting, which meant limitations of quality and quantity varied among patients. Meanwhile, we have altered AE treatment strategy when rituximab became a preferred choice for immunosuppression, causing only a small number of patients with low-dose rituximab combination enrolled for therapeutic protocol. Since randomized trials are difficult to conduct in rare diseases such as AE, real-world data might contribute to important information on treatment profiles and protocols. Although limited data on simplified regimen of combined optimal low-dose rituximab in our study, the result might be encouraging and presenting therapeutic implications for AE.

Altogether, in present study, the simplified regimen of combined low-dose rituximab (100 mg once) with common first-line therapy for AE with NSAbs, to our knowledge, firstly showed effective for short-term and long-term improvement, in parallel with reduced immunosuppressant and relapses, suggesting the advantages and benefits for combination of low-dose rituximab in the disease course. Moreover, the opportunity of rituximab protocol showed earlier initiation with better improvement, while frequency of first-line treatment had no influence on satisfactory outcome with rituximab combination. Our reports may expand therapeutic options and provide helpful references for NSAbs-associated AE, and further studies to corroborate these findings are warranted.