Antibody Therapies in Autoimmune Neuromuscular Junction Disorders: Approach to Myasthenic Crisis and Chronic Management

Abstract

Myasthenia gravis (MG) is a neurological autoimmune disorder characterized by muscle weakness and fatigue. It is a B cell–mediated disease caused by pathogenic antibodies directed against various components of the neuromuscular junction (NMJ). Despite the wide range of adverse effects, current treatment is still based on non-specific immunosuppression, particularly on long-term steroid usage. The increasing knowledge regarding the pathogenic mechanisms of MG has however allowed to create more target-specific therapies. A very attractive therapeutic approach is currently offered by monoclonal antibodies (mAbs), given their ability to specifically and effectively target different immunopathological pathways, such as the complement cascade, B cell–related cluster of differentiation (CD) proteins, and the human neonatal Fc receptor (FcRn). Up to now, eculizumab, a C5-directed mAb, has been approved for the treatment of generalized MG (gMG) and efgartigimod, a FcRn inhibitor, has just been approved by the U.S. Food and Drug Administration for the treatment of anti-acetylcholine receptor (AChR) antibody positive gMG. Other mAbs are currently under investigation with encouraging preliminary results, further enriching the new range of therapeutic possibilities for MG. This review article provides an overview of the present status of mAb-based therapies for MG, which offer an exciting promise for better outcomes by setting the basis of a precision medicine approach.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13311-022-01181-3.

Introduction

Myasthenia gravis (MG) is a rare autoantibody-mediated disease that targets the neuromuscular junction (NMJ). It is caused by the activation of B cells with subsequent production of autoantibodies directed towards different proteins of the postsynaptic endplate, which ultimately disrupt the normal neuromuscular transmission, leading to muscle weakness and fatigability. The main antigenic target is represented, in about 80–90% of cases, by the nicotinic acetylcholine receptor (AChR), followed by the muscle-specific tyrosine kinase (MUSK) and the lipoprotein-related protein (LRP4). Some patients however are defined as seronegative, meaning that no specific autoantibody has been detected [1]. Traditional therapies for MG include immunomodulation, in case of myasthenic crisis or acute worsening of MG symptoms, and non-specific immunosuppression. Notably, about 10–20% of patients remain refractory to immunosuppressive therapy [2]. This, together with the fact that corticosteroids and other immunosuppressive agents come with a wide range of often debilitating side effects, calls for an urgent need of new and more effective treatments for MG. As the knowledge of MG pathophysiology advances, so does the range of new potential therapeutic approaches. Specifically, monoclonal antibody (mAb) therapies hold great promise in delivering a more specific and effective therapy. Currently, various mAbs targeting different pathogenic pathways, such as B cell activation, T cell-B cell interaction, complement cascade activation, and immunoglobulin recycle, are under investigation. This review summarizes the rationale of mAb-based therapeutics in MG and its present status (Table 1).

Table 1.

Monoclonal antibody-based therapies for MG

Drug	Target	Study status	Administration	Clinicaltrials.gov number
B cell–directed mAbs
Rituximab	CD20	Phase III ongoing	IV	NCT02950155
Inebilizumab	CD19	Phase III ongoing	IV	NCT04524273
Tak-079	CD38	Phase II ongoing	SC	NCT04159805
Indirect B cell–targeting mAbs
Belimumab	BAFF	Phase II concluded	IV	NCT01480596
Iscalimab	CD40	Phase II concluded	IV	NCT02565576
Satralizumab	IL-6	Phase III not yet recruiting	SC	NCT04963270
Complement inhibiting mAbs
Eculizumab	C5	FDA approved for MG	IV	NCT00727194 NCT01997229
Ravulizumab	C5	Phase III ongoing	IV	NCT03920293
Zilucoplan	C5	Phase III ongoing	SC	NCT04115293
Pozelimab (+ Cemdisiran)	C5	Phase III not yet recruiting	SC	NCT05070858
FcRn inhibiting mAbs
Efgartigimod	FcRn	Expanded access	IV	NCT04777734
		Phase III ongoing	SC	NCT04735432 (placebo-controlled) NCT04818671 (OLE)
Rozanolixizumab	FcRn	Phase III ongoing	SC	NCT03971422 (placebo-controlled) NCT04650854 (OLE)
Nipocalimab	FcRn	Phase III	IV	NCT04951622
Batoclimab	FcRn	Phase II concluded	SC	NCT03863080

mAb monoclonal antibody, BAFF B cell–activating factor, FcRn neonatal Fc receptor, IV intravenous, SC subcutaneous

Myasthenia Gravis Pathophysiology

MG is one of the most studied and well-understood neurological autoimmune disorders. Several studies conducted on animal models of experimental autoimmune MG (EAMG) helped to better understand its pathophysiology [3] allowing to design new therapeutic approaches besides conventional therapy, as discussed further in this review.

MG patients are classified as early-onset (EOMG), when onset of symptoms is before age 50 years, and late-onset (LOMG), when the first onset of symptoms is after age 50 years [4]. EOMG and LOMG have very distinct epidemiological and pathological characteristics, which reflect different underlying disease mechanisms that probably stem from distinct genetic risk factors. In fact, different genetic variants within the major histocompatibility complex (MHC) locus contribute to EOMG and LOMG differences [5]. Other risk loci outside the MHC complex have been identified; for example, genetic variants near the TNIP1 gene are related to EOMG [6], while those close to the TNFRSF11A gene are associated with LOMG [7]. Recently, several novel genes, such as TRAF3, ORMDL3, and GSDMB, have been described as related to EOMG, further expanding the understanding of the different pathogenic pathways that underlie MG. [8].

Autoantibodies in MG

MG is the archetypal humoral-mediated autoimmune disease, characterized by different autoantibodies directed against various functionally important molecules at the postsynaptic membrane of the NMJ.

AChR antibodies are the most frequently identified antibodies, being detected in approximately 80–90% of MG patients, and are highly specific for disease. AChR antibodies are mainly of IgG1 and IgG3 subclass and are able to compromise the normal neuromuscular transmission through three main pathogenic pathways: (i) complement activation, which is the principal mechanism, with subsequent disruption of the synaptic folds on the postsynaptic membrane; (ii) crosslinking of AChR with successive endocytosis and degradation; and (iii) direct blocking of AChRs acetylcholine binding site [9]. MUSK antibodies are found in about 40% of anti-AChR antibody seronegative patients. Anti-MUSK antibodies are mainly of IgG4 subclass which have limited capability of activating the complement pathway [10]. Their pathogenic activity is carried through by interfering with normal AChR clustering and thus impairing neuromuscular transmission [11]. In about 19% of double anti-AChR and anti-MUSK seronegative patients, anti-LRP4 antibodies are found [12]. These antibodies interfere with MUSK activation by inhibiting the agrin-LRP4 interaction, which results in blocking of AChR clustering [13–15]. Moreover, anti-LRP4 antibodies are of IgG2 but also of IgG1 subclass and are therefore able to activate the complement cascade [16]. More recent studies have demonstrated the presence in triple seronegative patients of antibodies directed against agrin, a basal membrane protein essential for AChR clustering in its neural isoform (n-agrin). In mice models, it has been demonstrated that induction with anti-agrin antibodies is able to induce MG [15]. Other autoantibodies have been found in the serum of MG patients, mainly against collagen Q [17], rapsyn [18], ryanodine receptor [19], cortactin [20], titin [21], and the K_v1.4 voltage-gated potassium channel [22]. However, their potential pathogenetic role is yet to be proven.

T cells in MG

Before discussing the central role of B cells in MG pathology, it is necessary to underline the importance of T cell–B cell interaction in the pathogenesis of MG. Indeed, mature B cells require the action of CD4⁺ T helper cells to undergo somatic hypermutation and clonal selection, with subsequent development of follicular B cells in the secondary lymphoid organs [23]. Follicular B cells create germinal centers (GCs) and later differentiate either into long-lived memory cells, short-lived plasmablasts, or antibody-secreting plasma cells [23]. In Fig. 1, a schematic representation of the NMJ and of B cell-plasma cell differentiation is given.

Fig. 1.

Schematic representation of the terminal B cells lineage and antibodies involved in the autoimmune attack to the neuromuscular junction. B lymphocytes, plasma cells, and some of the key molecules involved in the immune activation are represented together with available monoclonal antibodies and biologicals targeting CD molecules or receptors. In bold, drugs are effective on different cells. MG, myasthenia gravis; NMJ, neuromuscular junction; MAC, membrane attack complex

Class switching and hypermutation of B cells in MG strongly depend on the activity of antigen-specific CD4⁺ cells [24]. Epitopes from each subunit, most commonly the α subunit, elicit an inflammatory response characterized by the production of IL-17 and IFN-γ by AChR-specific T cells, which supports the role of Th1 and Th17 cells in the pathogenesis of MG [25, 26]. Higher frequencies of Th1 and Th17 have also been encountered in MuSK MG, although very few studies have been conducted so far on this matter [27, 28]. The activity of T helper cells is generally counterbalanced by T regulatory (Treg) cells, which present a defective regulatory response in MG. Indeed, both thymic and peripheral Tregs have an impaired ability to suppress T cell responses [29–31]. This is further complicated by the fact that autoreactive T cells in the thymus are also resistant to Treg-mediated suppression [32]. A distinct subset of CD⁺ T cells, known as T follicular helper (Tfh) cells, has been recently described. Such cells are present in the lymphoid tissue and express the transcriptional factor Bcl6 and the chemokine receptor CXCR5, which allows them to migrate into the GC [33, 34]. Tfh cells are typically increased in the thymus of MG patients, and a higher frequency is reported not only in case of a thymic alteration, but in the normal thymus as well [34]. It is still debated whether Tfh cells exist in the blood. Numerous studies have demonstrated the presence of CXCR5⁺CD4⁺ cells in the periphery, possibly representing memory Tfh-like cells that migrated outside the GC [35–37]. These Tfh-like cells have been found to be particularly enriched in patients with AChR⁺ gMG (but not with ocular MG) compared to healthy controls [34]. However, it is still unclear if and how these cells contribute to MG pathogenesis. Interestingly, in MUSK⁺ MG the overall Tfh-like population is instead normal and comparable to healthy subjects [38]. Counterpart to Tfh cells are follicular regulatory T (Tfr) cells, which regulate Tfh cells activity by suppressing their interaction with B cells in the GC [39, 40]. The number and functionality of Tfr are also reduced in MG [34, 41].

Finally, another fundamental B cell–T cell interaction is represented by the binding of CD40 receptor, located on various antigen-presenting cells, including B cells, with its ligand CD40L, located on activated T cells. In fact, CD40-CD40L interaction induces Ig class switching, GC formation, and B cell differentiation into plasmablasts and plasma cells [42], making it an interesting therapeutic target, as later discussed.

B cells and Thymus Involvement in MG

The role of B cells in MG development has been largely assessed, starting with the identification of AChR IgG-producing B cells in the thymus of MG patients [43, 44]. Indeed, the thymus plays a crucial role in AChR⁺ MG, while it seems to not be involved in MUSK⁺ MG. Among AChR⁺ MG patients, about 80% have a thymic alteration, either thymic hyperplasia or thymoma. Thymic hyperplasia is the most common thymic abnormality, occurring in about 80% of cases. It is histologically characterized by an elevated number of GCs (which are absent in the normal thymus) with anti-AChR-producing B cells. B cell maturation and survival are then favored by a particular microenvironment granted by an upregulation in B cell-activating factor (BAFF) [45]. This evidence supports the theory for which the thymus is the site where immune tolerance to AChR is lost [46–48]. In fact, the thymus is where T cell selection takes place, specifically self-reactive T cells undergo negative selection in the thymic medulla, which is a pivotal process for central tolerance. Medullary thymic epithelial cells (TEC) express various tissue-specific antigens, including AChR, to present to T cells under the control of the autoimmune regulator (AIRE) [49, 50]. Dysregulation of the negative selection process results in an abnormal immune response towards various muscle proteins expressed on TECs, including AChR, titin, and ryanodine receptor [51]. Self-reactive T cells that elude central tolerance are normally eliminated by Treg cells, which are responsible for peripheral tolerance [36]. As previously mentioned, dysfunction of Treg and conventional cells is observed both in AChR⁺ and MUSK⁺ MG [52]. In thymoma, Treg dysfunction, lack of AIRE expression, and the absence of intratumor myoid cells all contribute to an altered T cell selection with defective self-tolerance. [53, 54].

Standard Treatments and Conventional Therapies

Immunosuppressive therapies in MG are aimed at contrasting autoimmune reactivity and, in particular, at counteracting the effect of anti-AChR antibodies at the NMJ. At present, we distinguish between acute phases of MG from the chronic course of the disease, which require a different therapeutic approach: immunomodulation for the former and immunosuppression for the latter.

Acute Phase of MG

Acute phases include rapid worsening of the disease reflecting a substantial modification of the MG patient clinical status. This condition may progressively lead, if not adequately treated, to the onset of a myasthenic crisis (MC) in which a respiratory insufficiency frequently occurs, necessitating mechanical ventilation with or without tracheostomy. Very rarely a MC can be the presenting symptom of the disease [55]. Infections are by far the most frequent cause of MC, followed by ab ingestis pneumonia. Another frequent cause of MC is erroneous administration of forbidden antibiotics, specifically fluoroquinolones and azithromycin [56–58], beta-blockers [58–60], and, more recently, checkpoint inhibitors [61]. Other drugs are currently under investigation for possible relation to de novo MG. In particular, alemtuzumab, an anti-CD52 mAb, induces an initial depletion of lymphocyte followed by a repopulation which can predispose to secondary autoimmunity. Such effect has been described for Graves’ disease [62] and immune-mediated thrombocytopenia and glomerulonephritis [63]. It has also been reported in a case of a patient with multiple sclerosis who developed AChR⁺ MG after the second dose of alemtuzumab [64]. However, the patient presented mildly elevated AChR titer before starting alemtuzumab; therefore, it is possible that the drug has unmasked an underlying MG.

Of note, as later discussed, treatment starting with corticosteroids may induce a MC, as transient worsening. Moreover, relevant emotional stress (e.g., surgery, grief) can induce a severe symptom worsening, if not a MC. About one-third of patients, however, do not have a clear-cut cause for a MC.

The therapeutic approach for an acute worsening of the disease is immunomodulation, also defined as rescue therapy, which consists of either therapeutic plasma exchange (TPE) or intravenous (i.v.) administration of high dose immunoglobulin (IVIg). The efficacy of both treatments is similar at day 15. The most recent Cochrane Review on IVIg for MG analyzed seven different randomized controlled studies on IVIg versus placebo, TPE, or no treatment in patients with MG [65]. Among those, two studies demonstrated no significant difference between IVIg and TPE in the treatment of exacerbations [66, 67]. One trial comparing IVIg to placebo demonstrated some evidence of IVIg efficacy in moderate or severe MG worsening [68]. Another trial comparing two doses (1 g/kg and 2 g/kg) for the treatment of MG showed no significant difference [69]. Another, but underpowered, study showed no difference in efficacy between IVIg and methylprednisolone [70]. IVIg treatment is usually performed with the following schedule of 0.4 gr/Kg body weight i.v. for 5 days. TPE effectively reduces levels of circulating IgG, including pathogenic autoantibodies. One plasma volume exchange is able to reduce immunoglobulin stores by 20% and serum immunoglobulin levels by 60%. Recovery of IgG levels back to baseline occurs about 6 weeks after the last course of TPE [71]. Frequency of plasma exchanges varies between different centers, as no definite protocol has been established. Generally, TPE is performed three times weekly for up to six exchanges, as more exchanges have limited evidence for benefit. In our center, we use a short standardized protocol of 2 procedures on alternate days with an exchange of 1 plasma volume for each procedure [72]. In case of clinical need, procedures can be repeated after 15 days. Despite clinical trials indicating, as previously mentioned, similar efficacy between IVIg and TPE in the treatment of MC, expert consensus suggests that TPE may be more effective also because its effect is more rapid [73]. Moreover, TPE may be more effective than IVIg in the treatment of MUSK⁺ MG patients. Compared to IVIg, TPE comes with greater risks linked to hemodynamic and venous access complications, which can however be minimized by using a peripheral access instead of a central one. The choice between IVIg and PLEX is also influenced by patient comorbidity. Specifically, TPE is contraindicated in sepsis, while IVIg cannot be used in hypercoagulability, renal failure, or immunoglobulin hypersensitivity [73].

At present IVIG and TPE are considered the gold standard for rescuing MG patients from clinical deterioration or MC, and no biological treatments, including mAbs, are currently employed for this purpose. However, considering the kinetic of clinical improvement (about 1 week) induced by eculizumab and efgartigimod, it would be conceivable to employ these drugs for a rescue therapy. Such an approach will need, however, a formal study to demonstrate that Eculizumab and efgartigimod will be at least comparable to IVIg or TPE to achieve a significant and rapid clinical improvement. Of note, it is advisable to eventually perform TPE before IVIg or mAb infusion, since TPE would remove the therapeutic immunoglobulins, leading to treatment failure.

Chronic Management

In order to guarantee a personalized treatment strategy, it is important to take into consideration various aspects, such as MG subgroup, associated antibodies, and thymic involvement, which can require different therapeutic approaches. On a general basis, chronic management of MG treatment is constituted by symptomatic treatment, steroidal and non-steroidal immunosuppression, and eventually thymectomy.

Symptomatic treatment is represented by anticholinesterases, and pyridostigmine bromide is the first-line of treatment in all forms of MG, except MUSK⁺ MG. Its use is however limited by frequent side effects like nausea, diarrhea, and increase in respiratory secretions, which are caused by the stimulation of muscarinic AChRs in the peripheral nervous system. It is also possible, with very high doses of pyridostigmine, to worsen MG due to polarization block [74, 75]. Prolonged therapeutic effect is rarely reached with anticholinesterases, and at the end, the majority of patients require add-on immunotherapy with corticosteroids or other immunosuppressants. Oral prednisone and prednisolone constitute first-line immunotherapy in MG thanks to their rapid effect. It is advisable to treat severe forms of MG with high steroid doses right away, since a higher dosage correlates with a more rapid response, generally within 2–4 weeks. In mild or moderate MG, it is possible to start at low doses and work up to the minimum effective dose [76, 77]. As aforementioned, it is important to keep in mind that initiation with steroidal therapy can transiently worsen MG symptoms; therefore, close monitoring is always advised when starting treatment, especially in case of bulbar weakness. Prevention of steroid-induced weakness can be achieved by treating with IVIg or TPE before starting steroid treatment, as stated by Sanders and colleagues in the international consensus guidance for management of MG [73]. Prolonged steroid treatment comes with many and often debilitating adverse events, such as osteoporosis, glaucoma, high arterial blood pressure, diabetes, weight gain, skin atrophy, and irritability [78]. To minimize such adverse events and to improve clinical stability, since there is a highly variable response to steroid monotherapy, it is recommended to combine steroids with other non-steroidal immunosuppressants. The latter include antimetabolites (like azathioprine, methotrexate, mycophenolate mofetil, or cyclophosphamide) and calcineurin inhibitors (like tacrolimus and cyclosporine). These immunosuppressants have a delayed response and can be used also in monotherapy. Since various months are often required to obtain a clinical effect, it is advisable, where possible, to do a steroid bridging until peak of efficacy is reached. Due to early toxicity, it is better to start at low doses and then taper to induction regimen. Medication choice is guided by various factors such as safety profile (which is higher for mycophenolate than cyclosporine or cyclophosphamide), child-bearing age (for which azathioprine is recommended), or unresponsiveness to other agents (which can justify treatment with cyclophosphamide) [79].

Considering that MG affects women twice as often as men, and generally in the child-bearing age, it is important to underline what are the therapeutic options for pregnant women. First-line treatment during pregnancy is represented by oral pyridostigmine with prednisone as the immunosuppressive drug of choice, since both have a good safety profile [73, 80, 81]. The main steroid-sparing immunosuppressive agent suitable for pregnant women is azathioprine, which, despite being associated with a higher rate of prematurity, low weight at birth, and intrauterine growth retardation, does not have any teratogenic effects [82]. However, while in Europe it is the non-steroidal drug of choice for MG in pregnancy, in the USA it is considered a risky drug due to case reports describing a higher risk of infections, anemia, thrombocytopenia, and leukopenia in babies exposed to azathioprine in utero [73, 83]. A possible mAb candidate for pregnant women is rituximab which, despite crossing the placenta, is not associated with significant adverse events on the fetus, except a temporary decreased B cell count [81, 84]. However, data collected on pregnancy is still very limited; therefore, rituximab should be considered only if the benefits on the mother outweigh the perceived risks on the fetus. MG crisis and severe worsening of MG symptoms can be treated with either IVIg or TPE with careful monitoring, specifically for symptoms related to hypovolemia in case of TPE or hyperviscosity in case of IVIg [73].

Generally, the therapeutic effect of non-steroidal immunosuppressant is often unpredictable, but clinical experience suggests a good response, around 70–80%, in the majority of cases, especially with mycophenolate mophetil, azathioprine, cyclosporine [85, 86], and tacrolimus [87]. This, however, does not necessarily correlate with the results from controlled studies.

Patients who do not respond or have contraindication to immunosuppression may benefit from chronic IVIg or PLEX therapy [88].

In thymomatous patients, removing of the tumor and surrounding thymic tissue is nearly almost necessary, although this often does not lead to clinical remission. In fact, the removal of the thymus, in thymomatous and non-thymomatous MG, reduces but does not eliminate AChR antibody levels, indicating that there is a stimulus outside the thymus that maintains antibody production [89]. As for AChR⁺ MG without thymoma, a multicenter, randomized, rater-blind clinical trial demonstrated that thymectomy is able to reduce MG severity and steroid dosage predominantly in EOMG [90, 91]. It is however important to underline that these studies compare the efficacy of thymectomy associated with prednisone versus prednisone alone. Indeed, thymectomy alone does not lead to clinical remission and its efficacy is appreciable mainly in a subgroup of MG patients (< 40 years of age and with thymic hyperplasia). Nonetheless, the current updated consensus for MG management recommends early thymectomy in non-thymomatous AChR⁺ generalized MG (gMG) patients, aged 18–50 years, to reach early clinical stabilization and minimize immunotherapy requirements and hospitalization. Current evidence does not support the indication of thymectomy in MUSK⁺ MG [88].

New Therapeutic Approaches Based on mAb Therapy

The increasing understanding of the biologic pathogenic pathways of MG has allowed in the latest years to conceive new drugs with a more targeted effect. In particular, the last decade has seen a wide range of mAbs being applied in MG with promising results.

Four types of monoclonal antibodies have been produced by antibody engineering [92]: (a) murine (-omab), entirely derived from a mouse hybridoma; (b) chimeric (-ximab) in which the variable regions are of murine origin whereas the constant regions are human; (c) humanized (-zumab) in which the molecule is from a human source except the domain binding to target is of murine origin; and (d) human (-umab) entirely derived from a human source. Murine and chimeric mAbs are the ones that may prone to intolerance. Specifically, immunomodulating mAbs can cause infusion-related reactions (IRRs), which usually occur within 30 to 120 min after start of mAb infusion, but can also present as a delayed reaction at up to 24 h. IRRs are generally mild to moderate and include the following: fever, chills, mild hypotension, rash, headache, and dyspnea. Severe reactions are rare and can include anaphylaxis, severe hypotension, and even cardiac arrest. It is therefore of utmost importance to closely supervise the patient during infusion, in order to eventually provide rapid emergency measures. As per the pathophysiology, IRRs related to mAbs are defined as cytokine-release reactions, due to the release of cytokines consequent to the binding of the mAb to the target cell. Some mAbs, including rituximab, can cause IRRs also by inducing an IgE-mediated allergic reaction, which is typically given by cytotoxic drugs [93–95].

Direct B cell Targeting mAbs

Current approaches of direct inhibition of B cells are mainly based on CD antigen-specific mAbs. The CD antigens are differently expressed depending on the subset of B cells and correlate to various functions like activation, inhibition, adhesion, and survival, thus representing an attractive therapeutic target for B cell–mediated diseases [96]. Therapeutic mAbs exert their cytotoxic effect through four different pathways: (i) direct apoptosis by blocking ligand binding of receptors essential for B cell survival, (ii) antibody-dependent cytotoxicity (CDC), (iii) antibody-dependent cellular phagocytosis (ADCP), and (iv) complement-dependent cytotoxicity (CDC) or cellular toxicity [97].

Rituximab, a chimeric IgG1 mAb-targeting CD20, is able to eliminate CD20-expressing cells either by complement-mediated or antibody-dependent cell-mediated cytotoxicity [98]. CD20 is expressed on the majority of B cells, but not on long-lived plasma cells and only on some plasmablasts, which are both responsible for AChR antibody production [99]. Therefore, it is expected for rituximab to be ineffective in reducing pathogenic AChR antibody levels, as confirmed by Díaz-Manera and colleagues [100]. On the contrary, another study demonstrated a statistically significant reduction of AChR antibody titers after treatment with rituximab [101]. A retrospective combined analysis evaluating previously published case reports of 169 AChR⁺ and MUSK⁺ MG patients, reported a clinical benefit from rituximab, with 72% of MUSK⁺ and 30% of AChR⁺ patients reaching minimal manifestation status or better [102]. A phase II randomized-controlled study of rituximab (beat-MG) enrolled 52 patients with non-thymomatous AChR⁺ gMG on a stable regimen of prednisone or prednisone plus another immunosuppressant agent. Primary outcome of steroid dose reduction was missed, and also, no significant changes in QMG or MG-Composite (MGC) were registered [103]. On the other hand, a prospective review on 54 MUSK⁺ patients revealed that the treated group experienced a clinical improvement and could be put on much lower doses of steroids and had a lower use of other immunosuppressants [104]. Taken together, these data indicate a differential response to rituximab among AChR⁺ and MUSK⁺ MG patients, with higher efficacy in MUSK⁺ patients. This could be explained by the fact that in MUSK⁺ MG the majority of autoantibodies is secreted by short-lived plasma cells, which, unlike long-lived ones, express CD20 [105, 106]. Current consensus guidance recommends early treatment with rituximab on MUSK⁺ patients who do not improve after initial immunosuppressive treatment. Dosing intervals for MG are four weekly doses of 375 mg/m², followed by a dose every 2 months. In refractory AChR⁺ gMG patients, rituximab is recommended only if other immunosuppressants resulted to be ineffective or scarcely tolerated, since its efficacy is uncertain [88]. There is an ongoing phase III, placebo-controlled, double-blind study evaluating the effect of rituximab in moderate-severe new-onset gMG. Patients must be naïve to immunosuppressants, with the exception of steroids (NCT02950155).

Other newer anti-CD20 mAbs have been engineered, but their potential efficacy in MG is unknown. Ocrelizumab and ofatumumab are human anti-CD20 mAbs, both with higher cytotoxicity for CD20 B cells than rituximab, that have demonstrated efficacy in the treatment of multiple sclerosis [107, 108]. Obinutuzumab acts by causing direct cell death, rather than complement-dependent cytotoxicity. It is yet to be investigated whether this translates into a higher efficacy, but for now, a case report on its use in a patient with refractory MG and lymphocytic leukemia showed its efficacy [109]. Considering the limit of CD20-targeting therapies in the treatment of AChR⁺ MG, newer molecules have been considered.

Inebilizumab is a humanized mAb which targets CD19-expressing B cells through antibody-dependent cell-mediated cytotoxicity. It is approved for the treatment of neuromyelitis optica spectrum disorders [110], and currently, a phase III randomized, double-blind, placebo-controlled study on AChR⁺ and MUSK⁺ gMG patients is ongoing (NCT04524273). Contrary to CD20, CD19 is expressed on a broader variety of B cells, such as pro-B cells, pre-B cells, and some plasmablasts and plasma cells. It is therefore expected that targeting CD19 may induce a greater decrease in antibody titers compared to anti-CD20 therapies.

TAK-079 is a fully human IgG1 mAb that targets CD38, which is expressed on various leucocytes subpopulations, mainly on plasma cells, plasmablasts, and natural killer cells, and is induced on activated T and B cells. TAK-079 depletes CD38⁺ cells by antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct apoptosis [111, 112]. Currently, a phase II, randomized, placebo-controlled study to evaluate safety, tolerability, and efficacy of TAK-079 is being conducted on AChR⁺ and MUSK⁺ gMG patients (NCT04159805).

Indirect B cell–Targeting mAbs

Belimumab is a humanized IgG1 mAb that targets and neutralizes the biologically active soluble B-lymphocyte stimulator, BAFF, and is currently approved for the treatment of systemic lupus erythematosus (SLE) [113]. Blocking of BAFF leads to reduced B cell differentiation, with subsequent reduction of CD19⁺ and CD20⁺ B cells, CD20⁺/CD69⁺ activated B cells, and CD27⁻ naïve B cells [114]. High levels of BAFF were observed in MG and in the hyperplastic thymus, which may contribute to altered B cell proliferation, survival, and antibody production. Moreover, polymorphisms in the BAFF gene correlate with MG susceptibility [115]. Belimumab has been explored as a therapeutic strategy in a phase II, placebo-controlled, multicenter, double-blind study on 40 AChR⁺ and MUSK⁺ refractory gMG patients. If eligible, participants were randomized 1:1 to receive either placebo or IV belimumab 10 mg/kg (weeks 0, 2, 4, 8, 12, 16, 20). Despite its encouraging results in SLE, in MG the study failed to meet its primary endpoint, which was a mean change in QMG from baseline at week 24 of follow-up [116]. Considering such results, belimumab may not hold much promise in the treatment of MG, in particular of AChR⁺ MG. On the other hand, further studies should be performed in MUSK⁺ MG, as the only two MUSK⁺ enrolled patients both fell under the placebo group, leaving no information on the potential efficacy of belimumab on the MUSK⁺ population.

As abovementioned, another appealing therapeutic target is CD40-CD40L inhibition, since the interaction between these two proteins leads to an overall increased immune response. A study conducted by Im and colleagues on an EAMG model highlighted that antibodies directed against CD40L were able to induce a reduction of proinflammatory cytokines [117].

Iscalimab (also known as CFZ533) is an anti-CD40 mAb that does not induce B cell depletion but rather acts by blocking primary and recall T cell-dependent antibody responses and also decreases germinal cell formation [118]. A phase II, multicenter, double-blind, placebo-controlled study investigating safety, tolerability, and efficacy of iscalimab in AChR⁺ and MUSK⁺ gMG patients has been completed. Results are only available on ClinicalTrials.gov website at the moment, but, despite good safety, primary endpoint of QMG change was not reached (NCT02565576).

Another subset of therapeutic mAbs has been engineered to target B cell activity via blockade of chemokines and cytokines. Indeed, proinflammatory chemokines and cytokines (ILs) play a pivotal role in the pathogenesis of various autoimmune disorders, including MG, as they are able to activate antigen-specific T-helper cells, B cells, and dendritic cells, and also promote plasma cells differentiation and development. An interesting target in this sense for MG is IL6, since it has been demonstrated to play a pivotal and damaging role in MG [119]. In fact, mice-lacking IL6, either from birth or later acquired, are resistant to MG. In addition, anti-IL6 has been demonstrated to be able to reduce autoantibody levels and contain the disease in a rat EAMG model [119, 120]. Similarly, in neuromyelitis optica (NMO) with anti-aquaporin 4 antibodies, it has been demonstrated that IL6, which is increased in the serum and cerebrospinal fluid of NMO patients, enhances the antibody production from plasmablasts as well as their survival, whereas the blockade of IL6 receptor (IL6-R) induced a dramatical decrease of plasmablast survival in vitro [121]. IL6-R blockade could therefore represent a promising therapeutic target for many immune disorders, including MG and NMO. Tocilizumab is a humanized mAb targeting both cell-surface-bound and soluble IL6 receptor (IL6-R). No trial on MG has been so far conducted, but a positive effect of tocilizumab has been described in two MG patients refractory to rituximab [122]. An evolution of tocilizumab, created by modifying the aminoacidic sequence in order to increase its half-life, is satralizumab, a human IgG2 mAb also targeting IL6-R. A phase III, multicenter, randomized, double-blind, placebo-controlled study evaluating safety, efficacy, pharmacokinetics, and pharmacodynamics of satralizumab in gMG is about to start. The study includes AChR⁺, MUSK⁺, and LRP4⁺ patients as well as adolescents (age ≥ 12 years) (NCT04963270).

Complement Inhibiting mAbs

As previously mentioned, complement activation constitutes the main pathogenic mechanism in MG. AChR antibodies bind the antigen and the subsequent binding of C1q to its Fc receptor activates the classical complement pathway which terminates with the formation of the membrane attack complex (MAC). The pore formed by the MAC on the NMJ causes focal lysis, with disruption of the post-synaptic folds and AChR loss [123]. A key point in the complement cascade is the formation of C5 convertase, which is essential for the initiation of the terminal pathway and the subsequent MAC formation. In fact, C5 represents the main target of current complement inhibitors [124].

Eculizumab (Soliris), a humanized anti-C5 mAb, was the first drug to be approved in the therapeutics of complement-mediated disorders, as it resulted to be effective in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) [125] and atypical hemolytic uremic syndrome (aHUS) [126]. Later studies also demonstrated its high efficacy in neuromyelitis optica spectrum disorder (NMOSD), as it dramatically reduced relapses [127]. In the REGAIN study, a multicenter phase III randomized, double-blind, placebo-controlled study, 125 non-thymomatous patients with refractory AChR⁺ gMG were randomized to placebo and eculizumab groups for a period of 26 weeks. Despite closely missing its primary endpoint, which was a 3-point reduction in MG-ADL score, the success in secondary endpoints, such as change in QMG and MG-QoL15 from baseline, was met. Among the responders, about two-thirds of patients actually experienced clinical improvement within 1 week, with many having a 5-point reduction in QMG [128]. Participants who completed the REGAIN study entered the open-label extension (OLE) study, which demonstrated a good safety profile and also a continuous benefit. Frequency of exacerbation was reduced by 75% (p = 0.0001) and 56% went into pharmacological remission or reached the state of minimal manifestations (MM). Clinical efficacy appeared in the first week after the first infusion and reached its peak around week 12 [129]. Data from the REGAIN study and its OLE indicated that eculizumab was able to significantly reduce fatigue [130], it worked well across muscle groups [131], induced a significant proportion of minimal symptoms expression [132], and led to rapid and sustained achievement of MM in patients with AChR⁺ refractory gMG [133]. Moreover, the long-term safety and efficacy of eculizumab in gMG were evaluated over a period of up to 3 years showing a good safety profile, and a significant reduction of the exacerbation rates, of the use of rescue therapy and of MG-related hospitalizations [134]. In 2017 FDA approved eculizumab for the treatment of AChR⁺ gMG and current guidelines recommend its application in severe refractory AChR⁺ gMG, although its role in MG treatment will probably evolve over time [88]. Dosing intervals of eculizumab in MG comprehend a loading dose of 900 mg/week for four doses and a maintenance dose of 1200 mg/every 2 weeks.

Ravulizumab (ultomiris) is another humanized recombinant mAb that binds C5 with high affinity, currently approved for PNH and aHUS. It has an enhanced pharmacodynamic and pharmacokinetic profile compared to eculizumab, as it has a considerably longer half-life, requiring IV dosing every 8 weeks (vs. every 2 weeks with eculizumab). Preliminary results of an ongoing phase III randomized, double-blind, placebo-controlled multicenter study demonstrate a good safety profile and a good efficacy, with immediate and sustained improvement in the MG-ADL and QMG scores (NCT03920293).

Zilucoplan is another complement inhibitor, which can be self-administered daily by subcutaneous injection. It is a small molecule which acts on two points of the complement cascade: it allosterically blocks C5 cleavage and directly inhibits the first step of MAC formation. Binding site of zilucoplan to C5 also seems to differ from that of eculizumab, as demonstrated by its ability to bind C5 in blood samples of genetically eculizumab-resistant patients. After promising results of a phase II double-blind placebo-controlled study [135], a phase III study began in 2019 and is currently ongoing (NCT04115293).

Pozelimab is a human IgG4 mAb which also targets C5 and is able to bind polymorphic C5 variations that eculizumab is unable to bind. A phase III, multicenter, randomized, double-blind, placebo-controlled study evaluating safety and efficacy of pozelimab combined with cemdisiran (a siRNA-targeting C5 mRNA) in gMG patients is about to start (NCT05070858).

It is important to underline that C5 inhibition increases the risk of Neisseria Meningitidis infection, and it is therefore mandatory to vaccinate with both B-serotype and quadrivalent vaccines at least 14 days prior to treatment start.

Neonatal Fc Receptor Inhibiting mAbs

Another important target for MG, besides CD, is the crystallizable neonatal FcRn, expressed on various cell types and intracellular vesicles. Importantly, it is expressed on the vascular endothelium where FcRn binds the Fc portion of the antibody and carries the IgG through the cell surface into the lysosome which ultimately releases it into the extracellular space [136]. This is a pivotal process to recycle and maintain circulating levels of IgG, thus representing an interesting therapeutic approach. Disruption of FcRn-IgG interaction, indeed, translates into catabolism of IgG with subsequent reduction of IgG serum levels. Targeting the FcRn using a mAb or a mAb-fragment has shown encouraging results in various autoimmune disorders, including MG.

Efgartigimod (ARGX-113) is a humanized IgG1-derived Fc fragment, which competitively inhibits the FcRn. Recently, a phase III multicenter, randomized, double-blind, placebo-controlled study to evaluate efficacy, safety, tolerability, quality of life, and impact on normal daily activities of efgartigimod has been completed [137]. Adult patients with an MG-ADL score ≥ 5 (> 50% non-ocular) could be enrolled, regardless of their serological profile. Primary endpoint was proportion of AChR⁺ MG patients with a sustained ≥ 2-point MG-ADL improvement for at least 4 weeks, in the first treatment cycle. One hundred sixty-seven patients were enrolled (84 in the efgartigimod group, 83 in the placebo group) and 129 (77%) were AChR⁺. Of these, 65 were enrolled in the efgartigimod group and 44 of them (68%) had a ≥ 2-point MG-ADL reduction in cycle 1, meeting primary endpoint, while only 19 (30%) of the 64 patients enrolled in the placebo had a similar response. Similarly, 41 AChR⁺ patients of the efgartigimod group were QMG-responders (≥ 3 point improvement for at least 4 weeks) in cycle 1, against 9 patients (14%) of the 64 placebo group. Generally, patients in the efgartigimod group experienced greater and sustained total mean score improvements in MG-ADL, QMG, MGC, and MG-QoL15r in cycle 1, with maximum improvement at week 4 for all measures, except MG-QoL15r, which maximally improved at week 5. Among those who received a second cycle, a higher number of patients in the efgartigimod group were MG-ADL responder (36 [71%] of 51) compared to the placebo group (11 [26%] of 43). Results in the overall population were comparable to those in the AChR⁺ population. Both in the AChR⁺ and in the AChR⁻ population, efgartigimod reduced IgG levels with each cycle. Similar to IgG, also AChR antibodies were reduced, and in both cases, the reduction corresponded to clinical improvement. Efgartigimod was well tolerated, with a good safety profile. The most common adverse events were headache and nasopharyngitis [137]. An open-label extension of this study (ADAPT +) is currently ongoing (NCT03770403). An expanded access of efgartigimod is available for gMG patients who are ineligible to participate in a clinical trial, before regulatory approval (NCT04777734). On December 17th 2021 the U.S. Food and Drug Administration (FDA) approved efgartigimod (now sold under the brand name Vyvgart) for the treatment of AchR⁺ gMG, making it the first-and-only FDA approved FcRn blocker. Dosing of efgartigimod for MG is 10 mg/kg IV four times per cycle (one infusion per week), eventually repeated according to patient symptoms, but not sooner than 8 weeks after initiation of the previous cycle.

Another study is currently ongoing with the purpose of evaluating the pharmacodynamic non-inferiority of efgartigimod administered subcutaneously (SC) versus intravenously (ADAPTsc; NCT04735432), and also an OLE study evaluating long-term safety and tolerability of SC efgartigimod is currently active (ADAPTsc + ; NCT04818671). A phase IIIb, open-label study (ADAPT NXT), investigating efficacy of different dosing regimens of intravenous efgartigimod in AChR⁺ gMG patients to maximize and maintain clinical benefit, is about to start (NCT04980495). Finally, a phase III, open-label, uncontrolled trial to investigate pharmacokinetics, pharmacodynamics, and safety of intravenous efgartigimod administered to children (above 2 and below 18 years of age) with gMG is also about to start (NCT04833894).

Rozanolixizumab (UCB7665) is a human anti-FcRn IgG4 mAb which has completed a phase II study including AChR⁺ and MUSK⁺ gMG patients [138]. Patients were initially randomized to receive SC 7 mg/kg rozanolixizumab or placebo weekly for three doses. After 4 weeks of monitoring, patients were randomized again to receive three weekly doses of SC 7 mg/kg or 4 mg/kg of study drug. Primary endpoint of at least a 3-point change in QMG was missed, while MG-ADL reduction was statistically significant, with over 50% of patients of the rozanolixizumab group experiencing at least a 3-point reduction, versus 10% of the placebo group. IgG and AChR antibody titers decreased by 68% at the end of the study. A phase III, randomized, placebo-controlled, double-blind trial is currently ongoing (NCT03971422), as well as the open-label extension (NCT04650854).

Nipocalimab (M281) is a fully human, recombinant anti-FcRn IgG1 mAb, which showed positive results, as reported by the company, in a phase II trial on AChR⁺ and MUSK⁺ gMG patients [139]. A phase III, randomized, placebo-controlled, double-blind study is currently ongoing (NCT04951622).

Batoclimab (RVT-1401), another anti-FcRn mAb, has completed a phase II trial on AChR⁺ gMG patients, but results are currently not available (NCT03863080).

Final Considerations

Eculizumab and efgartigimod have been demonstrated to be effective as add-on drugs to conventional immunosuppression through very specific mechanisms of action: blocking activation of the complement cascade at C5 level or rapidly increase of a highly selective degradation of IgG. Their positive effect in the clinical trials has led to new therapeutic options in the management of MG but has also introduced a series of crucial considerations. The first is to understand whether they can be used as a first-line immunosuppressive therapy, possibly allowing a modification of the current treatment algorithms. Such a hypothesis stemmed out from the very rapid clinical improvement observed upon exposure to both complement and FcRn inhibitors and from their reasonably good safety profiles. The rapid effect of both inhibitors may consent a safe verification of their immunosuppressive effect and keep respect of the ethical aspects, i.e., patients will not take the risk of a period of time lacking an effective treatment, as rescue therapies and conventional immunosuppression therapies can be introduced. Controlled clinical studies with head-to-head comparisons in myasthenic patients naïve to immunosuppression will possibly ascertain whether exposure to non-targeted immunosuppressive drugs such as corticosteroids or cytotoxic drugs can be avoided or reduced to non-risky dosages.

Interestingly, the very rapid clinical effect of eculizumab and efgartigimod, about 1 week observed in the REGAIN and ADAPT studies, suggests that these drugs may have a competitive effect with the current rescue therapies (TPE or IVIG) as their effect is seen on the average after 2 weeks. Of course, controlled studies are warranted also to check if this is applicable in the real-world setting.

Furthermore, other biologicals such as anti CD19, IL-6R, CD38, and CD40 will be tested as add-on therapies to conventional immunosuppressants in the next months and, in case of positive results, not only we will detect their efficacy profile, but we will also better understand the role of these molecules in the immunopathogenesis of the disease.

Prospectively, the use of antibody-targeted therapies in conjunction with mAbs targeting specific immune molecules will open a complete new therapeutic scenario which may abrogate or reduce to a minimal exposure the conventional drugs now in use. In this regard, a change in the clinical study protocols to search for the best drug association will be crucial, for example, by using adaptive studies or biomarker-guided trials. The introduction of highly specific therapies has already modified the therapeutic landscape of MG and an approach characterized by “precise” medicine is next door, and we do have to understand if it will be valid only for the long-term therapy or also for the acute conditions.

Supplementary Information

Below is the link to the electronic supplementary material.