Due to their essential role in the autoimmune response, B cells are the main candidate target cells for effective therapies in MG to inhibit autoimmunity. Biological drugs targeting B cells can be categorized into drugs that directly target B or plasma cells, drugs that block the survival and differentiation of B cells, and drugs targeting cytokines involved in the differentiation and activation ( Figure 1 ).

FcRn belongs to the heterogeneous family of MHC molecules, from which it differs for limited diversity and inability to present antigens. FcRn is a beta-2 macroglobulin-associated protein expressed in a wide variety of tissues, including epithelia, endothelia, intestinal cells, kidney, liver, and placenta (80). FcRn is critically implicated in the materno-fetal IgG transfer, a mechanism that protects offspring from infections in early life. The receptor continues to play a key immunological role beyond the neonatal period, being able to protect IgG from intracellular catabolism and recycle them back into the circulation. Intracellularly, the FcRn binds the Fc portion of IgG with high affinity within the pH acid microenvironment typical of endosomes, inhibits IgG lysosomal degradation and promotes their release outside the cells into the neutral pH circulation milieu (80, 81). This process, mainly occurring in the vascular endothelium, increases the IgG half-life, according to the specific binding affinity of different IgG isotypes to FcRn. IgG3 have been proven to have the lowest binding potential to the receptor, and indeed they have the lowest half-life in the circulation compared to other isotypes, likely as an effect of competition with IgG1 for FcRn (82).

FcRn is also widely expressed on the cell surface of several hematopoietic cells, including monocytes, macrophages, dendritic cells (DCs), neutrophils and B cells (83). Thus, the FcRn is involved in additional immune functions, other than IgG transport and recycling, including potentiation of innate immune responses to IgG forming immune complexes with their antigens, that is important for immune surveillance against infections (81). In neutrophils, the receptor enhances phagocytosis of IgG-opsonized bacteria (84). In DCs and other antigen presenting cells, the FcRn participates in the presentation/cross-presentation of antigenic peptides by MHC class II and I to CD4+ and CD8+ T cells, by directing the internalization, trafficking and processing of antigen-bound IgG immune complexes (81, 84). In this way, the receptor contributes to the activation of adaptive immune responses. It has been demonstrated that production of pro-inflammatory cytokines (e.g., IL-6, TNF- α) by innate immune cells in response to IgG immune complexes strictly depends on FcRn, since it requires IgG binding to FcRn, and can be inhibited by FcRn antagonists (85). Thereby, pharmacological FcRn inhibition can result in an anti-inflammatory action via mitigation of pathogenic inflammatory responses in the context of inflammatory and autoimmune conditions.

FcRn expression is modulated in different ways depending on the different immunological microenvironments: the exposure of human PBMCs to TNF-α, LPS or CpG is able to induce a significant increase of FcRn expression via NF-κB signaling pathways; contrariwise, activation of the JAK/STAT-1 signaling pathway by IFN-γ can down-regulate functional FcRn expression (86, 87). In addition, the promoter region of the FcRn-encoding gene, FCGRT, contains variable number of tandem repeats (VNTRs) that affect the receptor expression: subjects homozygous for VNTR3, which is the most common variant (~84% of human population), have higher FcRn expression, and increased IgG binding ability, than heterozigous subjects bearing the VNTR2 variant (VNTR2/VNTR3), which is the second most frequent variant (~13% of the human population) followed by VNTR1,4 and 5 (~3% of the human population) (88). These observations implies that the function of FcRn, and the magnitude of beneficial effects of its therapeutic blockage, could vary in different disease contexts and genetic backgrounds.

The role of the FcRn as regulator of IgG homeostasis has made it an attractive target for precision medicine in MG to reduce pathogenic antibody levels in a specific manner compared to plasmapheresis or immunoglobulins.

Preclinical studies showed safety and efficacy of an anti-FcRn monoclonal antibody, 1G3, in a passive and active model of EAMG: in rats in which MG was induced by passive transfer of anti-AChR antibodies, 1G3 treatment resulted in dose-dependent improvement of symptoms and reduction of pathogenic antibody levels in serum; in rats immunized with the AChR, the treatment significantly reduced the severity of the disease symptoms and the levels of both total IgG and anti-AChR antibodies (89). Similarly, in a mouse model of MuSK-MG, obtained by injections with purified MuSK-MG patients’ IgG4, FcRn blockade by efgartigimod reduced IgG4 levels and determined significant in vivo muscle function improvements, thus highlighting the potential of FcRn-targeted therapies to effectively improve MG (90).

Efgartigimod (ARGX-113) is the first FcRn antagonist approved (December 2021) in the USA by the US Food and Drug Administration (FDA). It is a humanized IgG1-derived Fc fragment able to bind FcRn with high affinity, thus competing with endogenous IgG and lowering their levels (91).

The phase 3 double-blind, multicentric randomized-controlled ADAPT study assessed the efficacy and safety of efgartigimod as add-on therapy in 167 patients with gMG on a stable dose of at least one IS treatment, including 129 (77%) AChR-MG patients, 6 (4%) MuSK-MG patients and 32 double (AChR/MuSK-) negative patients (92) ( Table 2 ). Efgartigimod (10 mg/kg) or matching placebo was administered as four infusions per cycle (one infusion per week), repeated as needed depending on clinical response no sooner than 8 weeks after initiation of the previous cycle. Both in the AChR-positive and -negative patients, IgG levels significantly decreased with each cycle. A significantly higher proportion of AChR-MG patients in the drug group were MG-Activities of Daily Living (MG-ADL) responders (68%) than in the placebo group (30%) after the first cycle. Moreover, in cycle 1 the drug led to sustained improvements of the total mean scores for MG-ADL, QMG, MG Composite (MGC), and MG Quality of Life 15-item Scale - Revised (MG-QoL15r) rating scales (92). In patients who received a second cycle, a greater proportion of patients in the drug group (71%) were MG-ADL responders compared with the placebo group (26%), with similar rates observed after cycle 1. The drug was well tolerated and the safety profile was good. The results in the AChR-negative patients were similar to those in the AChR-MG population. Efgartigimod effectiveness in inducing clinically meaningful improvement in MG-ADL was recently reported by Katyal and colleagues (93), who analyzed the treatment outcomes in a cohort of 37 AChR-MG patients, with all except one having completed at least one cycle and 28 patients having completed at least two cycles. Clinically meaningful improvement in MG-ADL was achieved in 72% (26/36) of patients after the first cycle, including 3 of 4 patients with thymoma, and 25% (7/28) of patients achieved minimal symptom expression status after the second cycle (93) ( Table 2 ). The long-term effect of efgartigimod along 14 months, and its impact on the disease course, was more recently reported in 19 MG patients (AChR-, MuSK-, LRP4-positive or triple negative MG). During the year before treatment 8 of 19 patients (42%) were hospitalized, and 15 of 19 (79%) needed treatment with plasma exchange or immunoglobulins; three of 19 (16%) were admitted to the intensive care unit. During efgartigimod, none of the patients was hospitalized and only one patient required plasma exchange and intravenous immunoglobulins (94) ( Table 2 ). Positive results on the real world use of efgartigimod in 36 seropositive and seronegative MG have been recently reported from Japan (95) ( Table 2 ). Long-term efgartigimod safety, tolerability, and efficacy has been confirmed by the OLE (up to 3-year extension) study of the ADAPT (96). Another FcRn blocker is Rozanolixizumab (UCB7665), a human IgG4 monoclonal antibody targeting the FcRn IgG binding region, whose safety and efficacy in gMG has been evaluated in a randomized, double-blind, placebo-controlled, adaptive phase 3 study (MycarinG) (97) ( Table 2 ). The study included 200 gMG patients, who received subcutaneous infusions once a week for 6 weeks of either rozanolixizumab 7 mg/kg (n=66), rozanolixizumab 10 mg/kg (n=67), or placebo (n=67). Both AChR- (n=179) and MuSK-positive patients (n=21), under conventional IS treatment, were included. Significant MG-ADL reductions from baseline were observed at day 43 (primary efficacy endpoint) for both the two rozanolixizumab groups compared to the placebo group. Improvements in MG-ADL, MGC, QMG, and Myasthenia Gravis Symptoms PRO (98) scores were observed as early as day 8 from the treatment start, the first time-point at which efficacy was assessed, and returned towards baseline levels by day 99, according with rapid reductions in total IgG at day 8 and their gradual increase by day 99. MuSK-MG patients had greater score reductions than the overall population, and all of them achieved an MG-ADL response (97). The MycarinG study results thus provided further support to safety and efficacy of FcRn inhibition for the treatment of gMG.

Nipocalimab (M281) is a fully human IgG1 monoclonal antibody with high affinity for the IgG binding site on FcRn. A phase 2 multicenter, randomized, double-blinded placebo trial (Vivacity-MG) assessed the nipocalimab efficacy in moderate-to-severe gMG (99) ( Table 2 ). The study included 68 MG patients randomly assigned to receive different intravenous doses of the drug (4 treatment groups) or placebo for 8 weeks. There were no treatment discontinuations due to side effects, and no difference between the treatment and placebo arms for adverse events (i.e. infections, headaches). A greater proportion of nipocalimab-treated patients exhibited rapid improvement (within two weeks of treatment) in MG‐ADL across all the 4 dosing arms compared to the placebo arm. The drug led to substantial reductions in serum total IgG and anti‐AChR autoantibodies that significantly correlated with MG‐ADL improvement (99). Safety and efficacy of nipocalimab is under investigation in an ongoing phase 3 multicenter study in adult patients with seropositive gMG (https://clinicaltrials.gov/study/NCT04951622).

Recently, Nowak and colleagues published the results of a Phase 2 proof-of-concept, randomized, double-blind, placebo-controlled trial with an OLE, on the efficacy of batoclimab, a subcutaneous fully human anti-FcRn monoclonal antibody, in AChR-positive gMG patients (100) ( Table 2 ). The drug was associated with significantly greater reductions in total IgG and anti-AChR antibodies from baseline to 6 weeks than placebo. MG-ADL, QMG, MGC, and MG-QoL15r scores showed improvements over time, but differences between the two treatment arms did not reach statistical significance, likely due to the small sample size (n=17) (100). A Phase 3 study performed in a higher number (n=132) of patients with gMG, including both AChR- and MuSK-positive patients (101), showed batoclimab ability to significantly increase the rate of sustained MG-ADL improvement, as early as week 2 for 4 or more consecutive weeks, with clinical effects and IgG reduction being similar to those previously reported for efgartigimod and rozanolixizumab. Currently, batoclimab therapeutic efficacy is under evaluation in another Phase 3 trial in AChR-positive gMG patients, expected to be completed in 2025 (https://clinicaltrials.gov/study/NCT05403541).

Anti-AChR autoantibodies belong to the IgG1 and IgG3 subclasses, that are the strongest activators of the complement system. Indeed, pathogenicity of these autoantibodies, but not that of anti-MuSK antibodies that belong to IgG4, is mainly due to complement fixation at the NMJ ultimately leading to postsynaptic membrane destruction and impairment of the neuromuscular transmission (102–104). Additional pathogenic mechanisms of anti-AChR antibodies are the direct functional block of the receptor and antigenic modulation, the last consisting in the cross-linking of two adjacent AChR molecules by the same antibody, causing AChR endocytosis and degradation (104).

The complement system, consisting of nearly 50 blood proteins, plays a key role in acute inflammation and defense against common pathogens as part of the innate immune response. Complement activation generates a robust proteolytic cascade that leads to opsonization for phagocytosis and osmotic/colloidal lysis of the pathogen, as well as generation of a potent inflammatory response through the production of pro-inflammatory molecules. Furthermore, the complement system acts to remove antigen-antibody complexes from circulation and is involved in adaptive immune responses, participating in the regulation of T and B cell activation (105).

In MG, complement-mediated damage of the NMJ occurs via the classical complement pathway, which starts with C1q binding to the Fc domain of the autoantibodies. Critical steps of the complement cascade are then the cleavage of C3 into C3a and C3b, and of C5 in C5a and C5b, the latter leading to the formation of the membrane attack complex (MAC, C5bC6C7C8C9 also known as C5b-9) that causes focal lysis of the post-synaptic membrane, disruption of post-junctional folds, and ultimately reduction of functional AChRs (102, 106, 107).

Several preclinical studies have demonstrated the role of the complement system in EAMG, as well as the efficacy of complement inhibition via recombinant proteins, chemicals, monoclonal antibodies and small interfering RNA (siRNA), in improving MG symptoms (108–113).

In muscle biopsies from MG patients, IgG and C3 deposition was localized at identical sites of post-synaptic membrane, and specifically on debris of junctional folds in the synaptic clefts and disintegrating junctional folds (114). Deposition of C9, the main component of the final and stable MAC, was also observed at the MG end plate regions, with the most intense depositions associated with the destroyed NMJs, indicating that NMJ loss may be largely due to complement activation (114).

Complement regulators are present on host cell surfaces to prevent autologous destruction by the complement system. Mice deficient for both the decay accelerating factor 1 (DAF1 or CD55), which inhibits C3 and C5 convertases, and the MAC inhibitory protein (MAC-IP or CD59), which inhibits MAC formation, developed a significantly worse EAMG associated with crisis, further supporting the role of complement role in MG (115).

Recently, Obaid and colleagues developed an assay based on the EK293T cell line modified to disrupt complement regulator gene expression via CRISPR/Cas9 genome editing, that was used to measure patient-specific autoantibody-mediated MAC formation by flow cytometry (116). By means of this system, they observed a subset of AChR-MG patients with high complement activity, who are likely patients in whom complement activation is the major disease mechanism, whereas the remaining patients showed low activity, reflecting possible complement-independent autoantibody-mediated mechanisms of NMJ damage (116). Circulating complement protein profile may be used as a biomarker for evaluating patient-specific complement activation: (i) Romi and colleagues found reduced C3 and C4 serum levels in AChR-MG patients with high autoantibody titers, indicating increased in vivo complement consumption (117); (ii) Liu and colleagues showed lower C3 levels in serum of AChR-positive compared to AChR-negative gMG patients and healthy controls (118), again suggesting complement activation-dependent C3 consumption; (iii) C2 and C5 levels were significantly reduced, and C3, C3b and C5a increased, in plasma of AChR-MG patients compared to controls in our recent study, suggesting “C2, C3, C5, C3b, C5a” as a profile suggesting complement activation (119); iv) increased levels of complement activation products (e.g. C5a, C3a, SC5b-9, C4a) were found in AChR-MG compared to controls by Staschei and colleagues (120), and v) lower serum C5 levels, indicating consumption, associated with higher sC5b-9 levels, were found in AChR-MG compared to patients with non-inflammatory neurological disorders by Ozawa and colleagues, further indicating complement activation in AChR-MG (121). Discrepancy among the results from the different studies could be due to differences in the clinical features of the patients’ cohorts (e.g. disease severity; IS treatments) or to technical issues related to the different methods used for the complement protein dosage, as well as to sample handling and storage and sample types (serum versus plasma). Recommendations for complement laboratory analyses should indeed be rigorously followed to avoid biased results, due to in vitro complement activation and consumption (122).

Since complement activation results from a cascade of multi-step events, many potential targets are available as candidates for therapeutic intervention.

C5 is a central component of the complement cascade as its cleavage into C5a and C5b is critical for MAC formation that is initiated by C5b (106). Biological drugs targeting C5 have emerged as a successful strategy to treat complement-mediated diseases, including MG (123).

Eculizumab is a fully humanized monoclonal antibody binding C5, approved by FDA for the treatment of AChR-MG patients with gMG (124). By inhibiting C5 cleavage into C5a and C5b, the drug is able to block the MAC formation and abrogate complement-mediated damage. Preclinical studies showed that administration of an anti-C5 monoclonal antibody in a passive immunization rat model not only restored strength in the animals, but also inhibited deposition of C9, thereby preventing endplate disruption (110). Eculizumab efficacy and safety in refractory AChR-positive gMG patients were demonstrated in the phase 3 REGAIN trial, and the following OLE study, demonstrating that eculizumab is able to elicit a rapid (i.e. by weak 1) and sustained (i.e. at least 130 weeks) improvement in muscle strength across ocular, bulbar, respiratory, and limb muscle groups and in daily activities (125–127) ( Table 3 ).

Ravulizumab is another FDA-approved human anti-C5 monoclonal antibody with similar mechanism of action of eculizumab but with a longer half-life, requiring less frequent infusions (every 8 weeks compared to 2 weeks for eculizumab). The phase 3 CHAMPION-MG study showed the drug ability to induce a clinically meaningful response in terms of significant MG-ADL and QMG reduction in AChR-MG gMG patients, with a rapid improvement within 1 week of treatment initiation in the treatment versus the placebo group, and adverse events rates were comparable between the two groups (128). The OLE phase of this study showed a sustained efficacy and long-term safety of ravulizumab, administered every 8 weeks, with rapid improvements in MG-ADL and QMG scores (within 2 weeks) and maintained up to 60 weeks (129) ( Table 3 ).

Zilucoplan is a macrocyclic peptide able to allosterically inhibit C5 cleavage. Compared to eculizumab and ravulizumb, that are administered intravenously, Zilucoplan offers the advantage to be administered subcutaneously at-home once daily by self-injections. Safety and efficacy of zilucoplan has been proven in patients with moderate to severe gMG in the phase III RAISE trial (130) ( Table 3 ). Indeed, MG patients assigned to zilucoplan showed a greater reduction in MG-ADL score from baseline to week 12, compared with those assigned to placebo, with incidences of serious adverse events and infections similar in both groups. An OLE study is ongoing (RAISE-XT, https://clinicaltrials.gov/study/NCT04225871).

Other drugs developed to block C5 for treatment of complement-mediated disorders are pozelimab and cemdisiran. The first is a fully human IgG4 monoclonal antibody against C5 with a proline substitution to promote stabilization of the disulfide bonds between the two heavy chains (IgG4P); the second is a small interfering N-acetylgalactosamine-conjugated ribonucleic acid (siRNA) which interferes with mRNA for C5, thus offering the opportunity to reduce C5 hepatic synthesis and hence circulating levels. The combination of pozelimab and cemdisiran was recently found to inhibit complement activity more efficiently (i.e. durable and more complete suppression) than the monotherapy with either pozelimab or cemdisiran in non-human primates, thus opening the way to clinical investigations on the potential of long-acting treatment with these two drugs for complement-mediated disease treatment (131). A phase 3 trial on the efficacy of a combination therapy based on intravenous pozelimab and subcutaneous cemdisiran is ongoing in gMG (NIMBLE, https://clinicaltrials.gov/study/NCT05070858).

Along with C5, additional complement cascade components are promising targets to counteract AChR-MG. Danicopan (ALXN2040) is an oral small molecule complement factor D (FD) inhibitor that showed improved benefit-risk profile as add-on therapy to ravulizumab or eculizumab in patients with complement-mediated paroxysmal nocturnal hemoglobinuria (PNH) compared to placebo (132). A phase 2 study on vemircopan, a similar oral factor D inhibitor molecule (ALXN2050) qualified for monotherapy in PNH, is ongoing in gMG patients (https://clinicaltrials.gov/search?intr=NCT05218096) ( Table 3 ). Since FD is essential for complement activation being necessary for the formation of the C3 convertase (107), its blockage is a promising approach to counteract the complement-mediated damage of autoantibodies and hence to treat AChR-MG.

Another oral inhibitor of complement activity is Iptacopan (LNP023), a small-molecule factor B inhibitor approved for PNH. The drug is able to block the activation of the alternative pathway at the level of the C3 convertase; indeed, factor B is a serine protease that drives the central amplification loop of this pathway (133). Inhibition of factor B has been proven to prevent complement activation in different complement-dependent diseases, thus sounding promising also for the treatment of MG.