Autoantibodies

Autoreactive B cells that escape peripheral tolerance checkpoint selection could target antigens of the CNS and cause autoimmune inflammation; however, no consistent B-cell antigen that is specific for MS and that causes demyelination has been identified to date despite numerous attempts. Intrathecal oligoclonal bands, a hallmark of MS diagnosis found in up to 95% of patients, ^{Reference Abdelhak, Hottenrott and Mayer27,Reference Thouvenot28} are not specific for MS (found also in e.g. meningitis and subacute sclerosing panencephalitis) and have been found to target intracellular antigens in patients with MS. ^{Reference Brändle, Obermeier and Senel29,Reference Willis, Stathopoulos, Chastre, Compton, Hafler and O’Connor30} In addition, detection of intrathecal IgM synthesis has been associated with onset of relapses and a more aggressive disease course. ^{Reference Villar, Masjuan and González-Porqué31} Similarly to antibodies of oligoclonal bands, B cells of MS lesions have been found to target intracellular antigens. ^{Reference Willis, Stathopoulos, Chastre, Compton, Hafler and O’Connor30} Antibodies previously thought to be present in MS such as antibodies against myelin oligodendrocyte glycoprotein (MOG) rather characterize a distinct disease entity (MOG-antibody disease) that encompasses pediatric acquired demyelinating syndrome, recurrent optic neuritis, acute disseminated encephalomyelitis, and neuromyelitis optica without anti-aquaporin four autoantibodies. A minority of MS patients harbor antibodies against a variety of antigens (some of them cell surface proteins) such as contactin-2, ^{Reference Boronat, Sepúlveda and Llufriu32} OMGP, ^{Reference Gerhards, Pfeffer and Lorenz33} and other peptide and lipid antigens. ^{Reference Yeste and Quintana34,Reference Kanter, Narayana and Ho35}

It must be noted that autoantibodies can also be responsible for the activation and chemotaxis of CD4+ T cells. The opsonization of myelin antigens, even at low concentrations, enhances the presentative competence of resident antigen-presenting cells (APCs), such as macrophages and dendritic cells, leading to increased recruitment of effector T cells and, consequently, aggravation of the disease severity, as explained in more detail below. ^{Reference Trotter, DeJong and Smith36–Reference Getahun, Dahlström, Wernersson and Heyman38}

B Cells as Antigen-Presenting Cells

B cells are efficient APCs and express MHC class II and costimulatory molecules, such as CD40, CD80, and CD86. ^{Reference Häusser-Kinzel and Weber25,Reference Kinzel and Weber39} They can capture soluble and membrane-tethered antigens via their B-cell receptor (BCR) and present them to T cells in an up-to-a 10.000 times more efficient way compared to myeloid APCs. ^{Reference Ancau, Berthele and Hemmer40} Evidence from experimental autoimmune encephalitis, a rodent model simulating "efferent" MS pathophysiology, opposes the hypothesis that the antigen-presenting function of B cells is central to the pathophysiology of MS. Specifically, MOG-specific B cells may initiate CNS inflammation and, consequently, the symptomatic onset of the disease, but do not affect either the proliferation or the molecular profile (i.e. secreted cytokines, activation markers) of MOG-specific T cells in the spleen and the draining lymph nodes. ^{Reference Flach, Litke and Strauss37}

On the other hand, a recently published report focusing on human leukocyte antigen (HLA)-DR15, which is the major genetic risk factor for MS, addresses how the immunopeptidomes presented by both DR15 allomorphs, DR2a and DR2b, on different APCs in the thymus, peripheral blood, and brain – including B cells – could affect autoimmune T cells. The results showed that DR2a and DR2b immunopeptidomes on B cells are significantly skewed toward HLA-DR-self peptides (HLA-DR-SPs) – compared to monocytes – which are consequently presented to autoreactive CD4⁺ T cells. These T cells responded robustly to individual and pooled HLA-DR-SPs in MS patients, compared to healthy donors, suggesting that DR2a and DR2b could jointly shape an autoreactive T-cell repertoire in MS. ^{Reference Wang, Jelcic and Mühlenbruch41}

B Cells as a Source of Cytokines

Physiologically, B cells can be a source of both proinflammatory and anti-inflammatory (regulatory) cytokines. B cells of RRMS patients however feature a profile that is skewed towards an abnormally hyperactive proinflammatory response. In mice, high levels of B-cell-secreted IL-6 can foster the differentiation of Th17 cells, while preventing the generation of T regulatory cells. ^{Reference Bettelli, Carrier and Gao42–Reference Korn, Mitsdoerffer and Croxford44} In MS patients, B-cell production of lymphotoxin alpha (LT-α), TNF-α, and granulocyte macrophage-colony stimulating factor (GM-CSF) appears elevated, forging a chronically inflammatory milieu within the CNS. ^{Reference Moreno Torres and García-Merino19,Reference Häusser-Kinzel and Weber25,Reference Li, Rezk and Miyazaki45} At the same time, anti-inflammatory, regulatory cytokines that are produced by B cells, such as IL-35, ^{Reference Shen, Roch and Lampropoulou46} but also TGF-β1 and IL-10, are instrumental in controlling inflammation in experimental models of MS. ^{Reference Arneth47}

EBV in MS

Among the infectious factors examined, the B-lymphotropic EBV has been shown to confer increased risk of developing MS, via, as yet, unclear mechanisms. ^{Reference Morandi, Jagessar, ‘t Hart and Gran48} Ninety-six percent of the general population is positive for IgG antibodies against EBV (indicating past infection), while in MS patients this percentage is almost 100%. ^{Reference Guan, Jakimovski, Ramanathan, Weinstock-Guttman and Zivadinov26} Moreover, a prospective cohort study of 955 incident MS patients showed a 97% of EBV (but not other viruses) seroconversion before development of the disease, significantly higher compared to controls. ^{Reference Kjetil, Marianna and H.B.49} Studies and experiments have shaped four major theories about EBV’s role in the pathogenesis of MS: the cross-reactivity hypothesis, ^{Reference Lang, Jacobsen and Ikemizu50} the bystander damage hypothesis, ^{Reference Angelini, Serafini and Piras51} the αβ-crystallin hypothesis, ^{Reference Pender and Burrows52} and the EBV-infected autoreactive B-cell hypothesis. ^{Reference Pender53} Cellular and CSF findings however only partially match the pathophysiology of MS as far as the first three hypotheses are concerned. One important finding is the recent demonstration of molecular mimicry between EBV transcription factor EBNA1 and CNS protein glial cell adhesion molecule (GlialCAM), leading to the production of cross-reactive antibodies with higher affinity towards an intracellular GlialCAM epitope. ^{Reference Lanz, Brewer and Ho54} The fourth hypothesis postulates that EBV-infected autoreactive B cells accumulate in the target organ and orchestrate the disease by producing antibodies and stimulating T cells due to a defect in their elimination by the antiviral CD8⁺ T cells. Moreover, the EBV antiapoptotic protein BHRF1, produced by both latently and lytically infected cells, inhibits B-cell apoptosis, ^{Reference McCarthy, Hazlewood, Huen, Rickinson and Williams55} resulting in immortalization of the autoreactive B cells it infects. In support of this hypothesis, substantial EBV persistence in B and plasma cells as well as meningeal B-cell lymphoid follicles of all MS cases examined was reported, ^{Reference Serafini, Rosicarelli and Franciotta56} but could not be reproduced in multiple independent replication studies. ^{Reference Sargsyan, Shearer and Ritchie57–Reference Torkildsen, Stansberg and Angelskår60} Overall, the epidemiological associations remain; however, no underlying biological mechanism has been consequently supported by experimental data.

Anti-CD20 mAbs

CD20 is a 33-37kDa transmembrane protein, which spans the membrane four times, thus consisting of two extracellular loops and intracellular C- and N-termini. Although some T cells with CD20 surface expression can be found in all lymphatic organs, are often CD8-positive, can be myelin-specific, ^{Reference Schuh, Berer and Mulazzani62–Reference Sabatino, Wilson, Calabresi, Hauser, Schneck and Zamvil64} and may correlate positively with disease severity, ^{Reference von Essen, Ammitzbøll and Hansen65} CD20 serves a more important role on B cells. The molecule is not expressed throughout the entirety of the B-cell line of differentiation, but only in pre-B cells and mature B cells, with stem cells and the majority of antibody-secreting cells being CD20-negative (Figure 1). ^{Reference Payandeh, Bahrami and Hoseinpoor66–Reference Tedder, Streuli, Schlossman and Saito68} Physiologically, CD20 plays a key role as regulator of calcium influx in the signaling pathways that lead to B-cell differentiation into antibody-secreting plasma cells ^{Reference Santos and Lima69} and its presence on the surface of most, but not all, B cells makes it an attractive target for monoclonal antibody-based therapy. B cells targeted by anti-CD20 monoclonal antibodies are eliminated via three main mechanisms: programmed cell death / apoptosis, complement-dependent cytotoxicity (CDC), or antibody-dependent cellular cytotoxicity (ADCC) processes. ^{Reference Maloney70} Evidence from animal studies shows that anti-CD20 antibody-mediated B-cell depletion may be incomplete in lymph node germinal centers. ^{Reference Schroder, Azimzadeh, Wu, Price, Atkinson and Pierson71} Moreover, cerebrospinal fluid B cells seem to be less affected than peripheral B cells by intravenous rituximab (the first anti-CD20 monoclonal antibody) administration, although the drug itself can be detected in a very low concentration (up to 1000 times lower than in the periphery) behind the BBB. ^{Reference Piccio, Naismith and Trinkaus72–Reference Monson, Cravens, Frohman, Hawker and Racke75} Of note, the limited access of anti-CD20 mAbs to the CNS due to their relatively high molecular weight could, at least to some extent, be overcome with intrathecal (IT) administration of anti-CD20 mAbs. The four main antibodies evaluated for anti-CD20 MS therapy are analyzed below.

Figure 1: Expression of cell surface antigens throughout B-cell maturation. CD19 is expressed in all stages of B-cell development, with the exception of stem cells and the majority of plasma cells. CD20 is not present on plasma cells, most plasmablasts, pro-B cells, and stem cells. BAFF-receptor (BAFF-R) is expressed on both immature and mature B cells in the germinal center, as well as memory B cells and late plasmablasts. Transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA) are expressed on germinal center B cells, memory cells, and antibody-secreting cells.

Anti-CD19 mAbs

CD19 belongs to the Ig superfamily and along with CD21, CD82, and CD225 contributes to the formation of a multimolecular signal-transduction complex that ultimately leads to the activation of PI-3 kinase. ^{Reference Tedder112} Compared to CD20, CD19 is expressed on B cells of earlier developmental stages as well as in more antibody-secreting cells and is thus an appealing therapeutic target (Figure 1). ^{Reference Chen, Gallagher, Monson, Herbst and Wang113} In addition to having a broader expression during B-cell stages of development and differentiation, CD19, unlike CD20, is selectively expressed on B cells and not T cells. ^{Reference Schuh, Berer and Mulazzani62} A phase 1 study assessing the pharmacokinetic (intravenous and subcutaneous) profile of inebilizumab, a humanized afucosylated IgG1κ anti-CD19 mAb, ^{Reference Herbst, Wang and Gallagher114} has been conducted in patients with relapsing MS with positive results, ¹¹⁵ but no phase III trials for MS are currently known to be underway.

Atacicept

Atacicept is a human recombinant fusion protein, consisting of a human IgG Fc portion and the extracellular domain of TACI receptor that binds both BAFF and a proliferation-inducing ligand (APRIL). ^{Reference Magliozzi, Marastoni and Calabrese116} The drug therefore competes for BAFF and APRIL binding with native TACI, which is both membrane-bound and soluble, ^{Reference Hoffmann, Kuhn and Laurent117} as well as, to a lesser extent, with the other receptors of the BAFF-APRIL system (BAFF-R and BCMA). ^{Reference Benson, Dillon and Castigli118,Reference Hartung and Kieseier119} After improving rheumatoid arthritis and systemic lupus erythematosus (SLE), ^{Reference van Vollenhoven, Kinnman, Vincent, Wax and Bathon120} atacicept was tried in MS.

Subcutaneous atacicept was evaluated in a 36-week, phase 2, double-blind, and placebo-controlled trial in 255 patients with relapsing MS. The trial was prematurely terminated when an increase in inflammatory disease activity was noticed despite immunoglobulin and naïve B-cell decrease, which led to the suspension of every atacicept trial in MS. ^{Reference Hartung and Kieseier119,Reference Kappos, Hartung and Freedman121,122} Another 36-week, phase 2, double-blind, and placebo-controlled atacicept trial in 34 patients with unilateral optic neuritis as clinical isolated syndrome also showed disease exacerbation, with a significantly higher proportion of patients converting to clinically definite MS compared with placebo. ^{Reference Sergott, Bennett and Rieckmann123} As atacicept effectively depletes naive B cells and induces a transient but marked increase in memory B cells (especially class-switched ones), ^{Reference Hoffmann, Kuhn and Laurent117,Reference Lühder and Gold124,Reference Jelcic, al Nimer and Wang125} possible reasons why atacicept aggravated MS include elimination of regulatory naïve B cells and enhancement of pathogenic memory B-cell function. ^{Reference Baker, Pryce, James, Schmierer and Giovannoni126,Reference Baker, Marta, Pryce, Giovannoni and Schmierer127}

Belimumab

Belimumab is a human IgG1λ recombinant monoclonal antibody directed against BAFF that prevents BAFF from interacting with its three receptors on the surface of B cells, thereby reducing B-cell survival, differentiation, and antibody production. ^{Reference Halpern, Lappin and Zanardi128,Reference Dubey, Handu, Dubey, Sharma, Sharma and Ahmed129} Interestingly, belimumab administration does not result in overt immunosuppression. ¹³⁰ While being moderately effective and FDA-approved for the treatment of SLE since 2011, it failed in myasthenia gravis, ^{Reference Hewett, Sanders and Grove131} a disease mediated by pathogenic autoantibodies. ^{Reference Stohl and Hilbert132} A phase 2, open-label trial of subcutaneous belimumab in addition to ocrelizumab (standard dose) in 40 patients with RRMS was scheduled to start within 2021. ¹³⁰

Evobrutinib

Evobrutinib is a small molecule drug that binds permanently to and deactivates Bruton’s Tyrosine Kinase (BTK). BTK is an integral part of the BCR signaling cascade that affects B-cell activation and is essential for B-cell maturation and their ultimate, terminal differentiation into memory or plasma cells. Of interest, BTK is involved in the entry of B cells into follicular structures. Knockout or absence of BTK results in lack of B-cell activation, moreover almost complete lack of peripheral B and plasma cells and low circulating immunoglobulin. ^{Reference Dingjan, Middendorp, Dahlenborg, Maas, Grosveld and Hendriks133–Reference Torke and Weber136} Importantly, about 75% of the CNS cells that express BTK are microglial, while BTK expression levels in the brain increase after demyelination. ^{Reference Martin, Aigrot and Grenningloh137} As evobrutinib can bypass the BBB and enter the CNS, it can affect microglial cells and B cells within the CNS.

Evobrutinib was the first BTK inhibitor (BTKI) to be tested as a monotherapy in relapsing MS. ^{Reference Becker, Martin and Mitchell138} In a double-blind, phase II trial (n = 267), evobrutinib was tested against placebo and dimethyl fumarate. The results showed that patients who received 75 mg of daily evobrutinib had significantly fewer GdELs during weeks 12 through 24 than those who received placebo (1.69 ± 4.69 against 3.85 ± 5.44, p = 0.005), while adverse effects were minimal (e.g. nasopharyngitis, alanine aminotransferase, and aspartate aminotransferase level elevation). ^{139,Reference Montalban, Arnold and Weber140} Evobrutinib is now being advanced to phase III evaluation, along with several other BTKI (some of them with reversible BTK binding); fenebrutinib, ibrutinib, and tolebrutinib. ¹⁴¹

While CD19/20 B-cell depletion has shown tremendous efficacy in reducing clinical and radiological MS activity, it raises several safety concerns on humoral deficiency with long-term usage in addition to a reduced response to vaccination. ^{Reference Luna, Alping and Burman86,Reference Nazi, Kelton and Larché142,Reference Bar-Or, Calkwood and Chognot143} These disadvantages could possibly be avoided with inhibition of B-cell activation and maturation with small molecules such as BTKIs. ^{Reference Torke and Weber136,Reference Dolgin144} In contrast with antibody-based B-cell depletion, BTKIs do not destroy or lastingly minimize the frequency of peripheral B cells, but seem to prevent the development of pathogenic B cells. ^{Reference Torke, Pretzsch and Häusler145} Their effect on disease activity does not seem to be as impressive as that of anti-B-cell antibodies, and they cannot control the pathogenic properties of B cells as rapidly; however, they are smaller in size, can penetrate the CNS, target microglia, and might therefore have a better effect on disability progression. ^{Reference Boschert, Crandall and Pereira146}