What's new in myasthenia gravis? - Autoantibodies in neurological disorders - Level 2 Amelia Evoli - the European Academy of ...

4rd Congress of the European Academy of Neurology

                    Lisbon, Portugal, June 16 - 19, 2018

                                 Teaching Course 16

       Autoantibodies in neurological disorders - Level 2

             What’s new in myasthenia gravis?
                                   Amelia Evoli
                                     Rome, Italy

Email: amelia.evoli@unicatt.it

Disclosure statement:
The author has no conflict of interest in relation to this manuscript

Myasthenia gravis (MG) is one of the best-characterized antibody-

mediated diseases, thanks to the accessibility of the neuromuscular

junction and the availability of effective animal models.

In MG, pathogenic auto-antibodies (Abs) cause morphological and

functional alterations of the postsynaptic membrane resulting in impaired

nerve-muscle transmission and fatigable muscle weakness. The result of

the Ab attack is a reduction of acetylcholine receptor (AChR) clustering,

which is crucial to the synapse efficiency and is regulated by a complex

network of core proteins, such as neural agrin, the muscle-specific

tyrosine kinase receptor (MuSK), and the low-density lipoprotein receptor-

related protein 4 (LRP4) (1). Both AChR and the MuSK-LRP4-agrin complex

are targets of the autoimmune response in MG, with differences in specific

IgG isotypes and pathogenic mechanisms.

MG is increasingly recognized as a syndrome more than a single disease, as

it   includes   several   subtypes   which   differ   in   terms   of   clinical

characteristics, prognosis and response to therapies.

Abs against AChR and MuSK cover together 90% of patients with MG, as

AChR Abs are detected in 80-85% and MuSK Abs in 5-8% of the whole

MG population (2). In these disease subtypes, genetic predisposition,

mechanisms of tolerance failure and Ab effects have extensively been

studied. As a rule, AChR and MuSK Abs are mutually exclusive (their

coexistence in the same patient is exceedingly rare) and are very specific

                                      1

for MG. Both are detected by radioimmunoassay (RIA) which is sensitive,

quantitative and largely available. Therefore, when MG is suspected on

the basis of clinical history and signs, AChR Abs are the first to be tested,

and MuSK Abs should be assayed in all AChR-negative patients.

In double seronegative (dSN) MG (i.e. MG with neither AChR nor MuSK Abs

on standard RIA), other Abs have been detected with different assays,

namely cell-based assays (CBAs). CBAs, which employ live transfected

cells, have not gained widespread use as they require specific skills and

equipment (3). Limited availability of serological testing can complicate

the diagnosis in dSN patients. On the other hand, methodological

differences make it difficult to establish the real prevalence of these new

Abs.

MG associated with AChR Abs

MG with Abs to AChR (AChR-MG) is characterized by a broad clinical

spectrum from isolated ocular symptoms to severe life-threatening

weakness, a bi-modal age at onset with female predominance in the early

onset population, and a high frequency of thymus alterations as thymic

follicular hyperplasia (TFH) and thymoma. AChR Ab positivity rate varies

according to the age at onset, from 60-70% in childhood to 97% in patients

older than 70 years, and to weakness extension, from 50% in ocular

myasthenia to 90% in generalized disease, and it approaches 100% in

thymoma patients (4-5).

AChR Abs are IgG1/IgG3 and induce MG through two main mechanisms:

complement-mediated focal lysis of postsynaptic membrane and increased

AChR internalization through cross-linking by bivalent IgG molecules

(antigenic modulation) (5).

                                     2

The muscle AChR is a pentameric glycoprotein made up of four subunits

(2a,1b,1g/e,1d); the fetal AChR (2a,b,g,d) is replaced by the adult

isoform (2a,b,e,d) by 33 weeks of gestation (6). A high proportion of AChR

Abs in patients’ serum target the a extracellular region and, particularly,

a conformational set of epitopes (the so-called main immunogenic region -

MIR) which includes the sequence 66-76 (7). Abs to MIR effectively induce

experimental autoimmune MG (EAMG) and show a closer correlation with

disease severity than the whole AChR Ab titer (7). Both in immunized

animals and in patients with MG, Abs against AChR cytoplasmic domains

and subunits other than the a subunit are commonly found, suggesting

that AChR released from the disrupted muscle membrane fosters epitope

spreading and provides a continuous source of antigen. While Abs to

cytoplasmic domains are not considered pathogenic, the presence of

extracellular epitopes, of other AChR regions, can enhance Ab-mediated

complement activation (8). In addition, Abs against the AChR fetal isoform

are potentially pathogenic during pregnancy. In fact, the presence in

myasthenic mothers, of Abs against the g-subunit, which disrupt the

function of embryonic AChR, can be responsible for the so-called “fetal

AChR inactivation syndrome” (FARIS). FARIS clinical features range from

lethal arthrogryposis multiplex congenita to mild myopathy predominantly

involving facial and bulbar muscles (9).   Diagnosis can be difficult (the

mothers can be completely asymptomatic) but is crucial, as in the absence

of adequate treatment the fetal disease recurs in subsequent pregnancies

(10).

AChR Ab effector mechanisms and specificities are targets of new

therapeutic options. Complement inhibition has proved effective in

generalized refractory AChR MG (11), in preliminary studies antigen-

specific immunoadsorption effectively depleted pathogenic Abs (12), a

                                    3

phase I trial of a therapeutic vaccine including peptides complementary to

the MIR has recently been completed (ClinicalTrials.gov identifier:

NCT02609022),    and   immunization       with   AChR   cytoplasmic   domains

prevented EAMG induction and suppressed the ongoing disease in mice (8).

AChR MG includes three main patient populations: early onset (age of

onset

Poly-autoimmunity is frequent in AChR-MG. The early-onset subtype holds

the highest association rate with other autoimmune diseases, with

endocrinopathies (namely thyroiditis) among the most common. On the

other hand, thymoma-MG is more typically associated with cutaneous,

hematologic and neurologic disorders. Among the latter, neuromyotonia

and autoimmune encephalitis are the most frequent (18).

AChR Ab production requires antigen-specific CD4+ T helper (Th) cells.

Contemporary studies have highlighted the role of Th17 cells in the loss of

tolerance to AChR (19), have shown an increased rate of circulating

follicular Th cells in generalized MG (20), and have reported defects of

both Treg (21), and B regulatory (Breg) cells (22). Longitudinal studies,

evaluating changes in these cell subsets in different MG phases and in

response to treatment, will clarify their role as markers of disease

activity.

It is well known that AChR Ab titer does not correlate with MG severity

and can still be positive many years after thymectomy and pharmacologic

treatment, in asymptomatic patients. AChR Abs are produced by plasma

cells (PCs), very likely long-lived PCs that are protected by their survival

niche against most immunosuppressants (23). While chronic Ab production

by long-lived PCs is thought to be involved in refractory MG development,

antigenic specificity and IgG isotype can contribute to Ab pathogenicity

and, ultimately, to symptom persistence and severity.

MG associated with MuSK Abs

MG with MuSK Abs (MuSK-MG) is characterized by a striking prevalence in

women and a focal weakness pattern with predominant involvement of

bulbar, neck and respiratory muscles. Ocular symptoms are milder and

                                     5

less asymmetrical than usually observed in MG patients and can be

overlooked. Lack of daily fluctuations, focal atrophy, unresponsiveness to

cholinesterase inhibitors and negative results of electrodiagnostic testing

in limb muscles can further complicate diagnosis. Thymus histology is

mostly normal-for-age with sparse lymphoid infiltrates and thymectomy

does not appear to improve the course of the disease. MuSK-MG responds

to immunosuppressive therapy (particularly to steroids) and most patients

experience sustained improvement with rituximab (24).

MuSK Abs are mostly IgG4, with smaller proportions of IgG1-3. Given to

their ability to exchange Fab-arms, IgG4 behave as bi-specific Abs and can

neither cross-link membrane-bound antigens nor activate complement.

Although MuSK IgG4 undergo Fab-arm exchange in vivo, these hybrid Abs

are pathogenic likely through direct interference with the protein function

(25).

MuSK extracellular region consists of three immunoglobulin-like (Ig)

domains and a cysteine-rich domain (CRD) (26). The MIR of MuSK is in the

Ig-1 domain (27), though Abs against Ig-2 and CRD have also been

described (28-29). As Ig-1 domain is crucial to MuSK-LRP4 interaction,

epitope-mapping studies support the view that MuSK Abs reduce MuSK

activation by interfering with LRP4 binding (30). Presynaptic defects and

Ab-mediated block of MuSK binding to ColQ (the collagen tail of

acetylcholinesterase) (31), can contribute to the disease pathogenesis.

In MuSK MG, epitope spreading is uncommon (27), and MuSK Ab titer

correlates with disease severity better than AChR Abs (32). Such a

correlation is even closer for MuSK-Ig1 Abs (27). The sustained decline of

MuSK Ab titer after B cell depletion suggests that short-lived rather than

long-lived PCs are the main Ab producers. A recent study has confirmed

                                    6

this view, showing that plasma blasts were indeed the main contributors

to MuSK Ab production (33). In addition, an increased frequency of CD4+T

cells producing inflammatory cytokines (34), and a decreased rate of Breg

cells have been reported (35) in these patients, mirroring the changes

observed in AChR-MG.

The association of MuSK-MG with IgG4-related disease has been reported

in a single case (36). The co-occurrence of other IgG4 Abs has not been

systematically investigated.

AChR and MuSK Ab detection by CBA

In CBA, AChRs are expressed on the surface of human embryonic kidney

cells, and clustered by co-expression with rapsyn as they are in vivo (37).

Serum Abs to “clustered AChR” have been detected in 16%-45.8% of dSN-

MG (38-40) patients often in association with juvenile and mild disease

(40). These Abs are mostly complement activating IgG1 and, in passive

transfer studies, reduced miniature end-plate potentials and decreased

AChR expression at mouse end-plates (37).

Recently, 8% of dSN-MG patients were found positive for MuSK Abs with a

highly specific CBA.    These patients were younger and less severely

affected than RIA-positive MuSK-MG patients (41). Collectively, these

studies show that AChR and MuSK Ab detection by CBA is specific and can

improve the diagnosis of MG, particularly in young patients with limited

forms of the disease.

                                    7

Abs to LRPA, agrin and intracellular antigens

Abs against LRP4 have been reported at varying rates, from 18.7% in a

multicenter analysis from Europe (42) to 1% in a recent survey from China

(43).

LRP4-immunized animals developed myasthenic weakness (44-45) and

LRP4 Abs (mostly IgG1 and IgG2) interfered with agrin binding and reduced

AChR clustering in vitro (46). While such effector mechanisms would

predict a severe phenotype, patients with Abs only to Lrp4 are

predominantly affected by mild disease (42-43, 47) with little evidence of

neuromuscular transmission impairment (48). Lrp4 Abs can co-occur with

AChR or MuSK Abs, more commonly with the latter (42-43, 46); these

double positive cases tend to have more severe symptoms (42).

Abs to agrin have been reported in 34 MG patients in different studies,

often in association with other disease-specific Abs, mostly AChR Abs (49-

51). Clinical features are not yet characterized. Sera from agrin-positive

patients inhibited MuSK phosphorylation and AChR clustering in myotubes

(49) and active immunization with N-agrin induced EAMG in mice (52).

Interestingly, LRP4 and agrin Abs have been found in patients with

amyotrophic lateral sclerosis at higher rates than in MG (53-54). These

reports need confirmation.

Abs to intracellular targets can be found in MG. Titin and ryanodine

receptor Abs (also called striational Abs) are detected in AChR-MG, are

strongly associated with thymoma and, to a lesser extent, with late-onset

MG and are markers of thymoma in patients with early-onset disease (55).

Cortactin is a tyrosine kinase substrate that acts downstream of

agrin/MuSK in promoting AChR clustering (56). Abs to cortactin were

                                    8

reported in 23% of dSN-MG and 9% of AChR-MG patients, mostly in

association with mild disease (57). Their role as biomarker is uncertain.

In summary:

   •   AChR and MuSK Abs are specific and well-characterized. These Abs

       identify two disease entities with distinct pathogenic mechanisms,

       clinical features and treatment responses

   •   CBAs have improved the serological diagnosis of MG. Multicenter

       studies and assay standardization are crucial to define the

       prevalence and clinical relevance of “new” Abs

   •   Abs to intracellular proteins are likely not pathogenic, are not

       diagnostic of MG but can provide clinically useful information.

                                     9

References

1.   Burden SJ, et al. Fundamental molecules and mechanisms for forming and
     maintaining neuromuscular synapses. Int J Mol Sci 2018; 19(2). pii: E490
2.   Gilhus NE, et al. Myasthenia gravis - Autoantibody characteristics and their
     implications for therapy. Nat Rev Neurol 2016; 12: 259-68
3.   Rodriguez Cruz PM, et al. Use of cell-based assays in myasthenia gravis and
     other antibody-mediated diseases. Exp Neurol 2015; 270: 66-71
4.   Chan KH, et al. Frequency of seronegativity in adult-acquired generalized
     myasthenia gravis. Muscle Nerve 2007; 36: 651-8
5.   Meriggioli MN, Sanders DB. Muscle autoantibodies in myasthenia gravis:
     beyond diagnosis? Expert Rev Clin Immunol 2012; 8: 427-38
6.   Hesselmans LF, et al. Development of innervation of skeletal muscle fibers in
     man: relation to acetylcholine receptors. Anat Rec 1993; 236: 553-62
7.   Luo J, et al. Main immunogenic region structure promotes binding of
     conformation-dependent myasthenia gravis autoantibodies, nicotinic
     acetylcholine receptor conformation maturation, and agonist sensitivity.
     J Neurosci 2009; 29: 13898-908
8.   Luo J, Lindstrom J. Acetylcholine receptor-specific immunosuppressive
     therapy of experimental autoimmune myasthenia gravis and myasthenia
     gravis. Ann N Y Acad Sci 2018; 1413: 76-81
9.   Hacohen Y, et al. Fetal acetylcholine receptor inactivation syndrome.
     A myopathy due to maternal antibodies. Neurol Neuroimmunol Neuroinflamm
     2015; 2: e57
10. Evoli A. Acquired myasthenia gravis in childhood. Curr Opin Neurol 2010; 23:
     536-40.
11. Howard JF Jr, et al. Safety and efficacy of eculizumab in anti-acetylcholine
     receptor antibody-positive refractory generalised myasthenia gravis (REGAIN):
     a phase 3, randomised, double-blind, placebo-controlled, multicentre study.
     Lancet Neurol 2017; 16: 976-86
12. Lazaridis K, et al. Antigen-specific apheresis of autoantibodies in myasthenia
     gravis. Ann N Y Acad Sci 2012; 1275: 7-12
13. Berrih-Aknin S, Le Panse R. Myasthenia gravis: a comprehensive review of
     immune dysregulation and etiological mechanisms. J Autoimmun 2014;
     52: 90-100
14. Cavalcante P, et al. Increased expression of Toll-like receptors 7 and 9 in
     myasthenia gravis thymus characterized by active Epstein-Barr virus infection.
     Immunobiology 2016; 221: 516-27
15. Kakalacheva K, et al. Intrathymic Epstein-Barr virus infection is not a
     prominent feature of myasthenia gravis. Ann Neurol 2011; 70: 508-14
16. Wolfe GI, et al. Randomized trial of thymectomy in myasthenia gravis. N Engl
     J Med 2016; 375: 511-22
17. Marx A, et al. The different roles of the thymus in the pathogenesis of the
     various myasthenia gravis subtypes. Autoimmun Rev 2013; 12: 875-84

                                        10

18. Evoli A, et al. Poly-autoimmunity in patients with myasthenia gravis:
     A single-center experience. Autoimmunity 2015; 48: 412-7
19. Cao Y, et al. Autoreactive T cells from patients with myasthenia gravis are
     characterized by elevated IL-17, IFN-γ, and GM-CSF and diminished IL-10
     production. J Immunol 2016; 196: 2075-84
20. Zhang CJ, et al. Augmentation of circulating follicular helper T cells and their
     impact on autoreactive B cells in myasthenia gravis. J Immunol 2016;
     197: 2610-7
21. Alahgholi-Hajibehzad M, et al. The role of T regulatory cells in immuno-
     pathogenesis of myasthenia gravis: implications for therapeutics. Expert Rev
     Clin Immunol 2015; 11: 859-70
22. Yi JS, et al. B10 Cell frequencies and suppressive capacity in myasthenia
     gravis are associated with disease severity. Front Neurol 2017; 8: 34
23. Mahévas M, et al. Long-lived plasma cells in autoimmunity: lessons from
     B-cell depleting therapy. Front Immunol 2013; 4: 494
24. Evoli A, Padua L. Diagnosis and therapy of myasthenia gravis with antibodies
     to muscle-specific kinase. Autoimmun Rev 2013; 12: 931-5
25. Koneczny I, et al. IgG4 autoantibodies against muscle-specific kinase undergo
     Fab-arm exchange in myasthenia gravis patients. J Autoimmun 2017;
     77: 104-15
26. Zong Y, Jin R. Structural mechanisms of the agrin-LRP4-MuSK signaling
     pathway in neuromuscular junction differentiation. Cell Mol Life Sci 2013; 70:
     3077-88
27. Huijbers MG, et al. Longitudinal epitope mapping in MuSK myasthenia gravis:
     implications for disease severity. J Neuroimmunol 2016; 291: 82-8
28. McConville J, et al. Detection and characterization of MuSK antibodies in
     seronegative myasthenia gravis. Ann Neurol 2004; 55: 580-4
29. Ohta K, et al. Clinical and experimental features of MuSK antibody positive
     MG in Japan. Eur J Neurol 2007;14: 1029-34
30. Huijbers MG, et al. MuSK IgG4 autoantibodies cause myasthenia gravis by
     inhibiting binding between MuSK and Lrp4. Proc Natl Acad Sci U S A 2013;
     110: 20783-8
31. Kawakami Y, et al. Anti-MuSK autoantibodies block binding of collagen Q to
     MuSK. Neurology 2011; 77: 1819-26
32. Bartoccioni E, et al. Anti-MuSK antibodies: correlation with myasthenia gravis
     severity. Neurology 2006; 67: 505-7
33. Stathopoulos P, et al. Autoantibody-producing plasmablasts after B cell
     depletion identified in muscle-specific kinase myasthenia gravis. JCI Insight
     2017; 2. pii: 94263
34. Yi JS, et al. Characterization of CD4 and CD8 T cell responses in MuSK
     myasthenia gravis. J Autoimmun 2014; 52: 130-8

                                         11

35. Guptill JT, et al. Characterization of B cells in muscle-specific kinase
     antibody myasthenia gravis. Neurol Neuroimmunol Neuroinflamm 2015;
     2: e77
36. Raibagkar P, et al. Is MuSK myasthenia gravis linked to IgG4-related disease? J
     Neuroimmunol 2017; 305: 82-3
37. Jacob S, et al. Presence and pathogenic relevance of antibodies to clustered
     acetylcholine receptor in ocular and generalized myasthenia gravis. Arch
     Neurol 2012; 69: 994-1001
38. Devic P, et al. Antibodies to clustered acetylcholine receptor: expanding the
     phenotype. Eur J Neurol 2014; 21: 130-4
39. Zhao G, et al. Clinical application of clustered-AChR for the detection of
     SNMG. Sci Rep 2015; 5: 10193
40. Rodríguez Cruz PM, et al. Clinical features and diagnostic usefulness of
     antibodies to clustered acetylcholine receptors in the diagnosis of
     seronegative myasthenia gravis. JAMA Neurol 2015; 72: 642-9
41. Huda S, et al. IgG-specific cell-based assay detects potentially pathogenic
     MuSK-Abs in seronegative MG. Neurol Neuroimmunol Neuroinflamm 2017;
     4: e357
42. Zisimopoulou P, et al. A comprehensive analysis of the epidemiology and
     clinical characteristics of anti-LRP4 in myasthenia gravis. J Autoimmun 2014;
     52: 139-45
43. Li Y, et al. Anti-LRP4 autoantibodies in Chinese patients with myasthenia
     gravis. Muscle Nerve 2017; 56: 938-42
44. Shen C, et al. Antibodies against low-density lipoprotein receptor-related
     protein 4 induce myasthenia gravis. J Clin Invest 2013; 123: 5190-202
45. Mori S, et al. Immunization of mice with LRP4 induces myasthenia similar to
     MuSK associated myasthenia gravis. Exp Neurol 2017; 297: 158-67
46. Zhang B, et al. Autoantibodies to lipoprotein-related protein 4 in patients
     with double-seronegative myasthenia gravis. Arch Neurol 2012; 69: 445-51
47. Marino M, et al. Flow cytofluorimetric analysis of anti-LRP4 (LDL receptor-
     related protein 4) autoantibodies in Italian patients with myasthenia gravis.
     PLoS One 2015; 10: e0135378
48. Nikolic AV, et al. Electrophysiological findings in patients with low density
     lipoprotein receptor related protein 4 positive myasthenia gravis. Eur J Neurol
     2016; 23:1635-41.
49. Zhang B, et al. Autoantibodies to agrin in myasthenia gravis patients. PLoS
     One 2014; 9: e91816
50. Gasperi C, et al. Anti-agrin autoantibodies in myasthenia gravis. Neurology
     2014; 82: 1976-83
51. Cossin J, et al. The search for new antigens in myasthenia gravis. Ann N Y
     Acad Sci 2012; 1275: 123-8
52. Yan M, et al. Induction of anti-agrin antibodies causes myasthenia gravis in
     mice. Neuroscience 2018; 373:113-21

                                        12

53. Tzartos JS, et al. LRP4 antibodies in serum and CSF from amyotrophic lateral
     sclerosis patients. Ann Clin Transl Neurol 2014; 1: 80-7
54. Rivner MH, et al. Agrin and low-density lipoprotein-related receptor protein 4
     antibodies in amyotrophic lateral sclerosis patients. Muscle Nerve 2017; 55:
     430-2
55. Skeie GO, et al. Titin and ryanodine receptor antibodies in myasthenia gravis.
     Acta Neurol Scand 2006; 113(S183): 19-23
56. Madhavan R, et al. The function of cortactin in the clustering of acetylcholine
     receptors at the vertebrate neuromuscular junction. PLosOne 2009; 4: e8478
57. Cortés-Vicente E, et al. Clinical characteristics of patients with double-
     seronegative myasthenia gravis and antibodies to cortactin. JAMA Neurol
     2016; 73: 1099-104

                                        13