Established and emerging immunological complications of biological therapeutics in multiple sclerosis

Running heading: Immunological complications of multiple sclerosis biologics

Babak Soleimani1

Katy Murray1

David Hunt1,2

1Anne Rowling Clinic, University of Edinburgh, Edinburgh, United Kingdom2MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom

Correspondence to David Hunt: david.hunt@igmm.ed.ac.uk

Abstract

Biologic immunotherapies have transformed the treatment landscape of multiple sclerosis. Such therapies include recombinant proteins (interferon-beta), as well as monoclonal antibodies (natalizumab, alemtuzumab, daclizumab, rituximab and ocrelizumab). Monoclonal antibodies show particular efficacy in the treatment of the inflammatory phase of multiple sclerosis.

However, the immunological perturbations caused by biologic therapies are associated with significant immunological adverse reactions. These include development of neutralising immunogenicity, secondary immunodeficiency and secondary autoimmunity. These complications can affect the balance of risks and benefits of biologic agents and 2018 saw the withdrawal from the market of daclizumab, an anti-CD25 monoclonal antibody, due to concerns about the development of severe, predictable autoimmunity. Here we review established and emerging risks associated with multiple sclerosis biologic agents, with an emphasis on their immunological adverse effects. We also discuss the specific challenges that multiple sclerosis biologics pose to drug safety systems, and the potential for improvements in safety frameworks.

Key Points The efficacy of biologic therapies for the treatment of multiple sclerosis continues to

increase This efficacy is balanced by serious adverse immunological events: both secondary

autoimmunity and immunodeficiency Biologic therapies pose unique challenges to multiple sclerosis pharmacovigilance

frameworks, with potential for improvement

1 Introduction: The landscape of biologic therapies in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) which is a major cause of acquired disability in young people[1]. While the exact immunological basis of MS remains elusive, its clinical course usually follows a highly distinctive pattern. Most patients develop episodes of transient neurological dysfunction which are often self-resolving[2]. These episodes are typically disseminated within the nervous system in both time and space[3]. This inflammatory phase is typically referred to as relapsing-remitting multiple sclerosis (RRMS) and its peak onset is seen in women in early adulthood[4]. In addition to this relapsing-remitting phase, most patients with MS develop gradual neurological dysfunction, which can evolve over years, in the absence of relapses. Progression can occur after a relapsing onset (secondary progressive multiple sclerosis, SPMS), or can develop from onset (primary progressive multiple sclerosis, PPMS).

Biologic therapies are complex drugs made in living cells[5]. In contrast to small molecule drugs, biologic agents such as recombinant proteins and monoclonal antibodies require multistep manufacturing processes such as cell culture, purification, stabilization, and packaging[6]. The advent of biologic agents for the treatment of MS in the 1990s signaled the arrival of a new era of disease-modifying therapies in MS - a disease previously considered refractory to immune therapy[7].

To date, 5 biologic agents have been granted licenses by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of RRMS (Figure 1). These biologics have a diverse range of effects (Figure 2), including targeted lymphocyte depletion (alemtuzumab, ocrelizumab), immunomodulation (interferon-beta, daclizumab – marketing authorization withdrawn March 2018[8, 9]) and impaired lymphocyte trafficking (natalizumab). The efficacy of some of these drugs is striking. For example, during treatment with ocrelizumab almost all radiological evidence of inflammatory activity can be suppressed during the relapsing-remitting phase of the disease[10, 11]. There is increasing evidence to suggest that aggressive targeting of inflammatory disease early in MS can improve long-term patient outcomes[12, 13].

Furthermore, the use of biologics extends beyond licensed indications. For example, rituximab, a B-cell depleting monoclonal antibody, being widely used to treat patients with MS based on efficacy data in both clinical trials and real-world use[14-16]. As such, the use of both approved and off-label biologics is increasing, particularly in the early stages of RRMS[17].

1.1 Immunological complications of biologic therapies for MSSuch therapeutic efficacy comes at a cost[18]. All biologic therapies used for the treatment of MS have been associated with serious adverse events – primarily related to the sequelae of injecting immunologically active proteins. A conceptual overview of immunological complications associated with biologics is given in figure 3. All recombinant proteins and monoclonal antibody therapies are immunogenic, leading to the development of neutralising antibodies. Typically, these antibodies reduce drug bioavailability, but in rare cases neutralising immunogenicity can itself be harmful[19]. Significant safety concerns have developed with the development of secondary immunodeficiency induced by these

drugs, perhaps best exemplified by progressive multifocal leukoencephalopathy (PML) resulting from natalizumab-associated focal CNS immunosuppression[20, 21]. In addition, these biologic agents are associated with the development of secondary autoimmunity, most notably autoimmune thyroid disease and idiopathic thrombocytopenic purpura (ITP) resulting from the use of alemtuzumab[22, 23]. Daclizumab, a monoclonal antibody directed against CD25 recently became the first licensed MS biologic agent to be permanently withdrawn from the world-wide market, serving as a strong reminder of the potential for serious and unpredictable immune consequences which can develop with these drugs in the postmarketing setting. Here we review the main immunological adverse events associated with biologic agents used to treat MS, using evidence from pre-clinical studies, post-marketing surveillance and case series. We also review wider drug safety issues posed by biologics.

2 Recombinant interferon-beta therapy

Recombinant interferon beta was the first biologic therapy approved for the treatment of MS[24, 25]. Interferon beta is an antiviral cytokine[26, 27] which exerts diverse immunomodulatory effects through its receptor, the interferon-α/β receptor (IFNAR) [27]. The rationale for developing this therapy for the treatment of MS was based on hypotheses of a viral aetiology or trigger for the disease[28, 29]. The mechanism of action remains unclear, but recombinant interferon-beta is a modestly effective drug which reduces relapses by about 30% [30, 31] with conflicting data on long-term benefits[32, 33]. Interferon-beta exerts its biological effects on many different cell types, leading to a broad range of biological effects in both the brain and beyond [27, 34, 35].

2.1 Neutralising antibody formationRecombinant protein therapies, like all biologic drugs, are immunogenic and induce significant titres of antibodies directed against the recombinant protein, a subset of which can neutralise the biologic effects of the therapy [36]. In the case of interferon-beta treatment, neutralising antibodies can occur in 10-25% of patients and are associated with a decrease in biological activity[37]. This can be detected as both a reduction of transcriptional response to interferon in the blood, and a reduction in therapeutic efficacy[38]. Methodologies for detecting and quantifying neutralising antibody formation vary considerably, making evaluation of the relative immunogenicity of different interferon preparations challenging. However cross-sectional studies suggest variability between preparations, with subcutaneous interferon beta-1b showing highest rates at 25-47%, followed by subcutaneous interferon beta-1a (Rebif) at 12-30% and intramuscular interferon beta-1a (Avonex) having lowest rates at 2-6%[37-39].

2.2 Secondary autoimmune diseaseMost commonly, type I interferon proteins induce self-limiting mild, systemic flu-like symptoms[40]. However, type I interferon proteins such as interferon-beta can induce autoimmunity[34, 41] and a number of autoimmune diseases have been described following the administration of recombinant interferon-beta[42], as well as interferon-alpha [43, 44]. Autoimmunity has predominantly manifested as liver and thyroid dysfunction[45, 46]. Thyroid dysfunction can occur as either thyrotoxicosis or hypothyroidism in 4% of cases[45]. Mild asymptomatic elevations in serum liver enzymes are common with interferon beta and have been observed in 37-67% of cases [43, 47-49] although more serious cases related to autoimmune hepatitis have been reported[43, 46, 50]. More rarely, case reports proposing a link between interferon beta to immune-mediated cutaneous diseases such as psoriasis, dermatomyositis and vitiligo have also emerged[51].

2.3 Immune-mediated thrombotic microangiopathyImportantly, interferon therapy has been considered a lower risk treatment compared to the newer generations of therapies for MS[17, 52]. However, few studies have assessed their safety in real-world practice, where patients are exposed to high levels of interferon over prolonged periods of time. A network meta-analysis of 2500 patients addressed this particular question, highlighting some of the previously unrecognised long-term real-world risks of interferon exposure. For example, in these studies, significant associations were identified with both stroke and depression in the setting of routine clinical use [53].

Another complication that has been linked to long-term exposure to type I interferon is thrombotic microangiopathy (TMA). TMA is a serious disease of the microvasculature which can be triggered by drugs, with a heterogeneous pathophysiological basis[54]. The primary manifestation of TMA is typically renal failure, but cerebral and cardiac microvascular beds can also be affected. TMA caused by recombinant interferon-beta is dose-dependent [55] and can develop in association with long-term treatment [55]. While the large majority of patients with interferon-associated TMA present in extremis to intensive care units with multiorgan dysfunction, they typically have a prodrome with detectable abnormalities of renal function and blood pressure[55]. In light of this the manufacturer has highlighted the potential for blood pressure and renal function monitoring[56]. An introduction of such a programme across Scotland was associated with early detection of cases, allowing interferon to be stopped before irreversible organ damage had occurred [55, 57]. In countries where efforts have been made to optimise case ascertainment, the incidence of TMA approaches 1 in 1000 patient-years[55].

Moreover, interferon-beta has been increasingly reported to induce nephrotic syndrome[58] through various underlying nephropathies including membranous nephropathy, minimal change disease[59], focal segmental glomerulosclerosis[60] and membranoproliferative glomerulonephritis[61]. These cases highlight the potential important of monitoring of renal function.

As market protections for recombinant interferon therapies come to an end, alternative ways of modifying interferon beta to improve aspects of drug delivery have been developed. Peginterferon b1a was developed by the addition of a polyethylene glycol chain to IFN-b1a, to improve its half-life and reduce the need for frequent injections. Pegylated interferon is injected weekly, rather than daily. The efficacy and safety of peginterferon b1a appear to be similar to other interferon-beta products[62], although it remains to be seen the degree to which it can be assumed that the safety profile of such “follow-on” biologics may differ from their originator, and this issue is discussed in more depth in the final section of this review.

3 Monoclonal antibody therapies

3.1 NatalizumabNatalizumab is a humanised monoclonal antibody directed against 4 integrins, both 4/1 and 4/7, which block lymphocyte trafficking into the brain [63]. This inhibition of lymphocyte transmigration leads to a significant reduction in relapse frequency in RRMS, and natalizumab was the first approved high-efficacy treatment [63-65], representing an important milestone for monoclonal antibody therapy for MS.

3.1.1 Neutralising antibody formationNeutralising antibodies develop in approximately 10% of patients treated with natalizumab [66]. They typically appear early in therapy, with the first infusions[67], and are associated with both infusion reactions and a loss of efficacy of the drug. Given that this drug is typically restricted to patients with severe forms of RRMS, this loss of efficacy can have serious and sometimes fatal consequences [68].

3.1.2 Secondary autoimmunityPerhaps surprisingly, natalizumab use in MS has not been clearly associated with the development of secondary autoimmunity. There has been a possible link to autoimmune hepatitis based on case reports of natalizumab associated hepatic failure where 6 out of 12 cases had features suggestive of autoimmune hepatitis on biopsy with associated antibodies[69, 70]. Consequently, hepatic monitoring is recommended[70]. Natalizumab use in Crohn’s disease has been linked with the development of other immune diseases such as sarcoidosis, but such problems have not been identified with its use in MS [71].

3.1.3 Focal immune suppression: natalizumab-associated PMLThrough its effects on inhibiting lymphocyte homing into the brain parenchyma, natalizumab causes a state of focal immunosuppression within the brain. Therefore, the identification of several cases of progressive multifocal leukoencephalopathy (PML) in clinical trials and the early post-marketing setting caused serious concern [20, 21]. Progressive multifocal leukoencephalopathy (PML) is a rare and often fatal demyelinating disease of the central nervous system caused by the John Cunningham Virus (JCV), which develops in patients with compromised immune systems. There is a high prevalence of JC virus exposure in both the general population and people with MS and it usually behaves as a harmless latent infection in immunocompetent individuals[72, 73]. PML had not been observed in prior trials of MS therapies, and is exceptionally rare as a spontaneous disease, so these early cases represented a strong safety signal. However, detailed evaluation of the level of the risk was complicated since both MS and PML are prototypical white matter diseases of the brain, and therefore can sometimes be difficult to distinguish. Market authorization of natalizumab was suspended in 2005 and a detailed assessment of patients exposed to the drug led to an initial estimate of PML in 1:1000 patients treated for a short period of time with natalizumab [21]. Given the high unmet need for patients with highly active relapsing remitting MS, and the particular efficacy of natalizumab in this subgroup, the drug was subsequently made available to patients with aggressive disease, in the context of a dedicated PML safety monitoring programme[21].

Extensive efforts have been made to mitigate PML risk with natalizumab, with the development of dedicated pharmacoepidemiological studies and registries and whose aim has been to stratify risk and prevent PML[74, 75]. Analyses of such registries has identified both length of exposure, JC virus antibody status and prior immunosuppressant use as important determinants of PML risk[74]. Furthermore, the titre of antibodies directed against JCV may play a role in stratification. High levels of antibody correlate with PML risk[76]. Patients who have all risk factors have a risk that is between 1:50-1:100[74, 76]. A recent pooled analysis from four large, observational, open-label studies has provided more accurate annual PML risks[76, 77]. The cumulative risk over 6 years for patients with all risk factors is approaching 2.5%. The exact methodology of calculating PML risk, and how best to communicate an individualized risk to patients, has been the subject of recent debate[78, 79].

Natalizumab is a high efficacy option and the risk of PML is low in patients who have no evidence of JC virus conversion[76]. However, false negative serum JCV antibody results can occur and patients can acquire infection during treatment[72]. The identification of patients at high risk of PML has been particularly important given the availability of different new therapeutic options. However, patients treated with natalizumab often suffer MS reactivation when the drug is stopped[63, 80]. Disease reactivation can be severe and sometimes even fatal, leading to a difficult clinical dilemma[81, 82]. There has therefore been intense focus as to how to reduce risk in such patients. At present, options include switching to other alternative therapies, for example fingolimod, alemtuzumab or rituximab[83, 84], or increasing the dosing interval from 4 to 6-8 weeks[85]. Optimal strategies for switching patients from natalizumab onto an alternative immune therapy remain to be determined, although studies suggest that B-cell depletion strategies following natalizumab are more effective than fingolimod at preventing disease reactivation. While these might represent promising strategies that balance the risk of disease reactivation against the risk of PML, systematic evidence is still needed to guide these difficult decisions. It is also clear that PML can manifest many months after natalizumab is cleared from the circulation[86]. As such, an “exit strategy” from natalizumab remains a complicated and poorly understood area.

3.1.4 Immune reconstitution syndrome and mitigating PML riskNotably, PML which develops in oncology patients treated with cell-depleting monoclonal antibodies carries a very high mortality [87]. However, this is not the case with natalizumab-associated PML in MS[88, 89]. The reasons for difference in clinical outcomes for PML between different biologic therapies remains poorly understood, but may relate to the ability of patients treated with natalizumab to permit relatively rapid immune reconstitution, which is an important predictor of PML outcome[77]. Therefore, a central tenet of risk mitigation has been the early identification of patients with PML, based on MRI screening, followed by rapid cessation of the drug. PML lesions can be detected on MRI scans before symptoms develop, sometimes months in advance[90, 91] and are often performed 3 monthly in high-risk patients[77]. Anecdotal reports suggest that such surveillance can lead to good outcomes, but systematic data are lacking[90, 92]. Early consideration had been given to rapid clearance of the drug from the circulation using plasma exchange upon PML diagnosis, but this approach is associated with an aggressive immune reconstitution syndrome (IRIS), leading to this practice being questioned[93, 94].

Indeed, it is not clear that any approach other than early identification of PML and prompt cessation of the drug has a role in the management of natalizumab-associated PML.

3.1.5 Other immunological complicationsHypersensitivity reactions are rare with natalizumab but have been described and variably manifest as urticaria-angioedema, anaphylaxis or delayed maculopapular eruptions[95-98].

Risk mitigation measures for natalizumab have been dominated by PML concerns, though it is clear that other CNS infections can occur, particularly with herpes viruses (e.g. herpes simplex virus 1 and varicella zoster virus)[99]. Concerns have also been raised surrounding signals relating to other consequences of prolonged CNS immunosuppression, such as CNS lymphoma[70, 100]. While natalizumab causes focal immune suppression, in both brain and gastrointestinal tract, signals with systemic infections have not been clearly identified, although reports of TB have been made[101]. Additionally, there are several case reports of a potential association of natalizumab with melanoma[70, 102, 103]. However, evidence of a causal relationship remains to be established[102, 103].

3.1.6 Combination therapiesThere have been few studies of combinations of biologic agents for the treatment of multiple sclerosis[104, 105]. The SENTINEL trial looked at combining natalizumab and interferon beta-1a[64]. It primarily compared interferon beta-1a alone to the combination of natalizumab and interferon beta-1a with results showing no significant difference in the incidence of immunogenic side effects such as infusion reactions or deranged liver enzymes[64]. Given the limited studies into combination therapies, it is difficult to conclude if combining biologics modulates overall risks, but more extensive trials may shed light on this further.

3.2 AlemtuzumabAlemtuzumab is a humanized monoclonal antibody which targets CD52 on lymphocytes and monocytes, causing prolonged T-cell depletion and complex modulation of the subsequent lymphocyte repertoire[106]. Originally developed to replace anti-lymphocyte globulin as a treatment for haematological malignancies[107], alemtuzumab has displayed efficacy in a variety of immune diseases[108, 109], with high efficacy seen in MS. The pivotal studies showed significantly improved efficacy with regards to relapse activity and MRI lesions compared to interferon[110, 111], with mixed results regarding disability progression in phase 3 trials[110], although additional studies are consistent with a durable effect on both clinical and imaging parameters[112-115]. In contrast to other biologic therapies which require regular injection, alemtuzumab is given in two cycles, one year apart, with long-lasting efficacy observed in the majority of treated individuals[110, 111].

3.2.1 Neutralising antibody formationAlemtuzumab is a highly immunogenic monoclonal antibody and neutralising antibodies develop in about 20-40% of patients[116]. It is not clear the degree to which this can lead to a reduction of efficacy of the drug, though it has been suggested that this can interfere with the ability of the drug to cause lymphocyte depletion[116]. In particular, it is not clear whether the development of these antibodies may interfere with cycles of treatment

beyond the second yearly cycle, or whether novel tolerizing strategies might reduce the incidence of neutralising immunogenicity[117].

3.2.2 Cytokine release SyndromeEarly reports of Campath-1H (the original name of alemtuzumab, reflecting its development in the pathology laboratories of Cambridge University) usage showed the development of a moderately severe infusion reaction in almost all treated patients, which was associated with widespread cytokine release and sometimes associated with acute neurological deterioration[118-120]. In one case report, the cytokine release was associated with acute pneumonitis and pericarditis[121]. The use of adjunctive high dose corticosteroids appear to have mitigated the severity of this response, although reactions including rashes and pyrexia remain very common but manageable[120].

3.2.3 Secondary autoimmunityAlemtuzumab is a cell-depleting monoclonal antibody which leads to a rapid and profound lymphopenia[107]. Lymphocyte reconstitution is a prolonged process and lasts months to years, and is associated with a spectrum of clinical manifestations, most typically as autoimmunity[122]. The development of secondary autoimmunity has been an important factor in the safety of alemtuzumab and has been identified since the earliest studies of its use in people with MS[123]. In initial clinical studies in MS, approximately 30% of treated patients developed a spectrum of secondary autoimmune disease, most commonly affecting the thyroid, but also causing more serious autoimmune diseases such as ITP and Goodpasture’s disease[12, 124]. Detailed clinical and scientific surveillance of secondary autoimmunity has illuminated not only risk mitigation strategies, but also provided insights into fundamental mechanisms of autoimmunity arising in the context of immune reconstitution[124-126].

Studies of lymphocyte repopulation have shown that T-cell recovery after alemtuzumab is driven by homeostatic proliferation of memory-like CD4 and CD8 cells, which can produce pro-inflammatory cytokines[122, 126, 127]. There is no clear association between the level of lymphopenia and autoimmunity[127], but individuals who develop autoimmunity show reduced thymic production and generate a more restricted T-cell repertoire[128]. However, as yet there are no predictive tests which facilitate identification of cases at higher risk of these complications[125].

Taken as a whole, the development of secondary autoimmunity develops in the five years following first treatment with alemtuzumab and arises most frequently between 12 and 18 months [129]. Secondary autoimmune disease encompasses a broad spectrum of endocrine, renal, haematological and dermatological complications, described in detail below.

Thyroid autoimmunity: The development of Graves’ disease was reported early in the clinical development of alemtuzumab for MS[123]. In a prospective study of 248 patients treated with alemtuzumab, 20% of patients developed thyroid autoimmunity, with Graves’ disease as the most common clinical phenotype, followed by a transient thyroiditis[129]. In these cases, there was an initial thyrotoxic phase, followed by normalisation of thyroid function or return to normal function. Regular thyroid function testing is recommended from baseline, before treatment, till 48 months after discontinuation[130-132]. A significant

majority of cases required medical treatment, with about a quarter of Graves’ disease cases requiring radioactive iodine. Cases of thyroid cancer have also been reported, although it is not clear as to whether this represents an artefact of enhanced thyroid monitoring[133].

Idiopathic thrombocytopenic purpura (ITP): ITP is the second most commonly observed autoimmune disease associated with alemtuzumab[129]. A death from ITP, due to intracranial haemorrhage, occurred in the context of phase II clinical trials for alemtuzumab for MS[12], highlighting both the potentially very serious nature of this condition and the need for risk mitigation. ITP develops in approximately 3% of cases and typically responds to conventional therapy without the need for long-term treatment [22]. Onset of illness was typically abrupt, with a limited role for haematological screening. Early recognition of symptoms helped initiate prompt treatment in most cases, highlighting an important role for patient education in conjunction with screening[12, 22, 129]. Current suggestions for screening are monthly full blood counts from baseline to 48 months after the last course, with platelet counts <100x109/L prompting repeated testing and urgent referral to haematology if persistent or associated with new onset bleeding[130-132].

Goodpasture’s Syndrome: Alemtuzumab is associated with renal immune-mediated adverse events in approximately 0.5% of patients. The best described complication is anti-glomerular basement membrane disease (Goodpasture’s syndrome)[23], but membranous nephropathy has also been reported[134]. Although rare, both can be associated with poor renal outcomes, including end-stage renal failure, but these outcomes can be mitigated with early initiation of appropriate therapy. Currently, suggested monitoring consists of measuring renal function and urinalysis at baseline and monthly thereafter[131, 132], but some centers have developed more specific guidance regarding what actions to take in the presence of serum creatinine elevations, proteinuria and/or haematuria[130, 134]. The presence of haematuria and significant serum creatinine elevations should always warrant immediate further assessment[130, 134].

Autoimmune skin disorders: Case reports have linked alemtuzumab with both vitiligo[135] and alopecia areata[136], both of which are T-cell mediated autoimmune disorders. Although seemingly rare, these disorders may be under-recognised and patients may need to be warned about potentially experiencing these side effects[136].

3.2.4 Secondary immunodeficiencyIt is perhaps surprising that people with MS who are treated with alemtuzumab are relatively immunocompetent in the years following their pulsed treatment, given the prolonged CD4 lymphopenia which results[137]. However, while uncommon, opportunistic infections do occur and can be serious. There is a clear risk of listeria meningitis which appears maximal around the time of drug infusion[138, 139]. This has been reported in about 0.1% of treated patients[138]. Although this usually responds to appropriate antimicrobial treatment, listeriosis in this context can be fatal[138]. Listeria meningitis can cause brainstem symptoms, mimicking a relapse, emphasizing the careful need for detailed evaluation of new neurological events while receiving treatment. The temporal predictability around the time of infusion makes antibiotic prophylaxis an option, as currently advocated in the UK, together with exclusion of listeria-prone foods for one month following treatment. Furthermore, Listeria monocytogenes has an incubation period of up to

70 days and this can be further prolonged with corticosteroid use, which is commonly used prior to alemtuzumab infusions, therefore putting patients at higher risk of colonisation. Consequently, patients may be best advised to avoid listeria-containing foods for a more prolonged period of time before and after infusions[138].

More recently, there have been reports of opportunistic pulmonary infection with Nocardia beijingensis[140], Pneumocystis jirovecii and cytomegalovirus[141], suggesting that although still rare, atypical and opportunistic infections may still occur, highlighting a need for an awareness of such complications, particularly around the time of pulsed therapy. Furthermore, associated viral infection with human papilloma virus leading to cervical dysplasia has also been described, making it mandatory for women to undergo annual screening in Europe[131, 142].

3.2.5 Emerging signalsGiven the breadth of serious adverse immunological events with alemtuzumab, it is perhaps not surprising that a number of serious adverse events have been detected later in the post-marketing setting. In particular, a recent signal for acalculous cholecystitis has been detected[143], a case of serum sickness-like reaction[144] and two recent cases of hemophagocytic lymphohistiocytosis (HLH) have been described[145]. There have also been reports of thyroid papillary cancer and melanoma[111, 114], raising concerns for increased risk of malignancy in treated patients, although the exact degree of risk remains unclear. Additionally, there have been cases of severely exacerbated CNS inflammation following alemtuzumab initiation, which have responded to rituximab and plasmapheresis, suggestive of severe B-cell mediated autoimmunity [146]. Finally, a recent analysis of data from the FDA adverse event reporting system found that alemtuzumab had a higher than expected odds ratio for the development liver injury[49].

3.2.6 Predicting autoimmunity after alemtuzumabAt present there is no clinically useful marker which can be used to predict autoimmunity following treatment with alemtuzumab. Studies have implicated a potential role for IL-21 in driving this secondary autoimmunity and, since IL-21 can be genetically predetermined, provides a potential insight into how pharmacogenetics and immunophenotyping of patient might in the future play an important role in personalized therapies[125].

3.3 DaclizumabDaclizumab is a humanized monoclonal antibody, directed against the alpha subunit of the IL2 receptor (CD25)[147]. This drug was initially developed as an immunosuppressant to prevent renal transplant rejection, but was subsequently developed for long-term subcutaneous administration, with less antibody-dependent cytotoxicity than earlier forms of the biologic agent. This resulting formulation is known as daclizumab high-yield process (DAC-HYP)[148]. The mechanism of action of daclizumab remains unclear and, while it was initially developed to interfere with IL-2 signaling through its high affinity receptor, the presence of CD25 on many critical immune cell subsets, including regulatory T cells, suggests a potentially broad set of anti-inflammatory and pro-inflammatory activities[149].

The pivotal trials of daclizumab showed significant efficacy compared to interferon-beta[148, 150]. However, in 2018, daclizumab was withdrawn from the worldwide market, due to a concern about a new specific signal of autoimmune encephalitis, but more broadly on the background of a concerning and growing risk of severe, unpredictable and fatal autoimmune disease affecting both liver and brain[9].

3.3.1 Severe secondary autoimmune diseaseIn initial studies, cutaneous adverse effects were reported in 37% of DAC-HYP patients and although most were mild-moderate in severity, there were more severe immunogenic cutaneous reactions such as toxic skin eruptions or drug reaction with eosinophiliaand systemic symptoms (DRESS)[151]. Hepatoxicity also was identified as a concern for daclizumab during clinical trials[148], with hepatic adverse events occurring in 16% of patients, significantly higher than those treated with recombinant interferon-beta. Most of these were mild or moderate elevations in serum transaminase levels. However, two fatal liver events subsequently developed, one in clinical trials and the other early in the post-marketing setting[152]. While the exact nature of these cases remains unclear, it is likely they had an autoimmune basis, were unpredictable and occurred despite stringent hepatic monitoring. In light of these events daclizumab’s use was severely restricted.

3.3.2 Severe secondary CNS autoimmunityShortly after these concerns regarding serious hepatotoxicity, a series of cases with autoimmune encephalopathies, were reported, again including fatal cases. Review of cases referred to a German center demonstrates significant heterogeneity. Some encephalitides were associated with anti-N-methyl-D-aspartate (NMDA) receptor antibodies and anti-glial fibrillary acidic protein (GFAP) antibodies, while other demonstrated evidence of CNS vasculitis and overlap with eosinophilic inflammation[153, 154]. Antibody-mediated encephalopathies have also been reported months after stopping treatment, suggesting vigilance for secondary autoimmunity may need to be extended for many months after drug cessation.

At present the mechanistic basis of these complications can only be speculated. It will be important to understand whether, perhaps due to an effect on targeting regulatory T cells[155], patients treated with daclizumab lost an important “immunological brake”, causing what might have otherwise been mild and self-limiting immune reactions to be catastrophic and severe.

3.4 Ocrelizumab and other B-cell depleting monoclonal antibodiesCD20 is expressed on most B cells and monoclonal antibodies such as ocrelizumab, ofatumumab and rituximab are targeted against CD20 and work to produce a rapid, but transient depletion of CD20 expressing B cells in the peripheral circulation[15]. Following the success of anti-CD20 cell depleting monoclonal antibodies in phase 2 clinical trials[11, 15], fully humanized antibodies have been developed (ocrelizumab, ofatumumab), with strong anti-inflammatory activity displayed in phase 3 clinical trials, including evidence for the first time of efficacy in progressive forms of MS[10, 156]. To date, ocrelizumab is the only licensed anti-CD20 monoclonal antibody licensed for MS.

Rituximab has been widely used as therapy for both malignant and autoimmune conditions and has shown significant promise as disease modifying therapy for MS[15]. While it is not yet a licensed treatment for MS, it is widely available in many countries and there is significant experience with its use in the treatment of MS.

3.4.1 Neutralising antibodiesAnti-B cell therapies are typically given every 6 months and can be associated with significant infusions reactions, including anaphylaxis and serum sickness[157]. Ocrelizumab has been associated with neutralising antibodies in 0.9% patients[158], whereas anti-rituximab antibodies have been detected in 37% of relapsing-remitting MS patients and 26% of progressive MS patients treated with this drug[159].

3.4.2 PMLPML is known to be associated with rituximab therapy[87] in the context of oncological disease, and outcomes from this are universally poor, with a 90% mortality rate. It is therefore little surprise that a case associated with ocrelizumab has been reported in which a 55 year old female with MS developed PML shortly after switching to ocrelizumab from natalizumab[160]. Consequently, at the present time, the risk of PML with anti-B cell monoclonal antibodies remains to be clarified and may be difficult to interpret since these drugs may be used as a way of switching patients at high risk of PML off natalizumab[83] [87].

3.4.3 HypogammaglobulinaemiaPlasma cells do not express CD20, hence it was previously considered that antibody-mediated immunity would be preserved despite rituximab-induced B cell depletion. However there have been increasing reports of rituximab causing hypogammaglobulinemia and antibody deficiency across a variety of conditions including MS and related neuroinflammatory disorders[161]. Moreover, the resulting hypogammaglobulinemia has been reported to develop both after a single dose or repeated doses and can be either transient or persistent in nature[162]. Most immunoglobulin subclasses are affected and patients are often found to have suppressed antibodies to previous vaccines such as pneumococcus and tetanus[161, 162]. Patients have also been found to be unable to mount sufficient humoral responses to vaccinations once hypogammaglobulinemia has been induced. Moreover, there have been suggested predisposing risk factors which include previous or concomitant immunosuppressive treatment; receiving rituximab for malignant disease; having low immunoglobulin levels prior to treatment or prolonged rituximab treatment[163].

The main complication of hypogammaglobulinemia is the risk of infection. Respiratory tract infections make up most of associated infections, but reports have also included other serious infections such as PML secondary to reactive JC virus, reactivated hepatitis B and cytomegalovirus[164]. In cases of severe and/or recurrent infections intravenous immunoglobulin (IVIg) has been used [161, 165].

3.4.4 MalignancyWhile rituximab has been used extensively for the treatment of haematological and inflammatory diseases, ocrelizumab use has been largely restricted as yet to the clinical trial

setting. Within this setting signals for cancer, such as breast cancer, have been identified, but not yet fully evaluated [10, 156].

4 Challenges for biologic safety in MS

4.1 Capturing the long-term risks of immunomodulationA major unresolved issue, affecting all MS therapies, is the accurate evaluation of their long-term risk-benefit profile. Drug approvals are typically given after large phase 3 pivotal trials which typically last 2-3 years [10, 166], but MS is a lifelong illness, with disease-modifying drugs sometimes required for decades[2]. While such trials allow quantification of the positive and negative effects of these drugs over a short period of time, they do not adequately inform the long-term risks and benefit required for day-to-day decision making in the clinic[2]. Most monoclonal antibody therapies for MS exhibit high efficacy and can suppress inflammatory activity on brain scans by up to 95%[10]. What is less clear is how durable these effects are, and how they translate into meaningful long-term outcomes such as prevention of progressive disease. Attempts have been made to study the long-term efficacy of recombinant interferon therapies (together with glatiramer acetate, a complex non-biological drug), however study outcomes appear highly dependent on the methodology used[32, 33, 167, 168]. Moreover, such studies have focused on efficacy rather than safety [32], and have provided no data as to the long-term safety of interferon therapies and played no role in the detection of late-emerging complications[53, 55].

A major focus of safety concern with immunotherapies typically relate to morbidity arising from both serious infection and cancer, which are associated with chronic states of immunosuppression. For example, safety signals regarding cancer have been raised in phase 3 studies of ocrelizumab, but will require more detailed post-marketing studies to clarify these signals[10]. At present there are few robust long-term studies of biologic safety which are adequately powered to address these concerns.

4.2 Sensitivity to manufacturing changeBiologic therapies in multiple sclerosis pose an additional and unique set of drug safety challenges. This stems from their molecular complexity and the potential for their biological properties to change, for better or worse, with manufacturing. For example, the manufacture of biologic agents requires multistep manufacturing processes such as cell culture, purification, stabilisation, and packaging[6]. Such processes can change over time, either as a result of natural variability (“drift”), or by design (“evolution”)[5]. Through this process of incremental change, established brands can undergo up to 50 changes in the way they are made[169], with each change undergoing limited, if any, clinical testing. For example, a recombinant protein might undergo sequential change in the cell clone to make the protein, a change in stabilizer and a change in packaging. This raises the theoretical possibility that the risk-benefit profile of the drug might alter over time. Such changes are sometimes actively marketed as “reformulation”, but more often than not the prescriber and patient will be unaware of these changes[5].

An example of how such “reformulation” can affect positive and negative parameters of a drug is demonstrated with recombinant interferon-beta[57]. TMA was recognized as a complication of interferon-beta late in the lifetime of the drug, over a decade after it was approved and widely used. Independent observations made by different groups reported an increase in cases coinciding with the introduction of a reformulated version of an approved

interferon-beta [57, 170, 171]. This increase was noted simultaneously in multiple countries and was specific for the reformulated product. Interestingly, the reformulated interferon product was of the same in vitro strength and potency[172], but was associated with significantly fewer neutralising antibodies[173]. Despite the same in vitro potency, the reformulated drug displayed higher bioavailability, which may reflect an unmasking of toxicity caused by the planned reduction in immunogenicity[174]. Therefore, the intended improvement of safety through the reduction of drug immunogenicity may have had an unintentional consequence of increasing bioavailable dose. This is important in addressing which changes in a drug require closer monitoring and more extensive clinical testing, highlighting a critical role for immunogenicity in mediating adverse event profile. Indeed, similar observations were made with recombinant erythropoietin, where an increase in pure red cell aplasia, mediated by anti- erythropoietin antibodies, was observed with a reformulation of higher immunogenicity [19]. It may be that reformulations which change a drug parameter – in particular its immunogenicity – require closer safety observation than those where the drug is unchanged, and effectively comparable.

4.3 Follow-on and biosimilar products“Biologic evolution” represents one way that an established branded drug can change, perhaps imperceptively, and in a way that is not apparent to the prescriber and patient. However, when market exclusivity for a biologic ends, it is possible for follow-on biologics to be developed, such as biosimilar drugs. Biosimilars are biological products with no clinically meaningful structural differences from the originator/branded biologic. Biosimilar medicines are important since they represent a potential route to reduction in the costs of drugs, which are increasingly problematic[175]. Biosimilar products provide their own set of pharmacovigilance challenges and regulatory approaches have differed, with the first biosimilars approved only recently in the US, whereas European regulation has approved such medicines for many years [176]. As market exclusivity for many of the biologics described above ends, this will place further challenges on the drug safety framework. These multiple challenges highlight the need for robust pharmacovigilance studies that are powered and designed to detect long-term risks of drug exposure, together with capacity to follow and track manufacturing changes. Given the increasingly availability and complexity of biologic therapies in the treatment of MS, it would seem an opportune time to evaluate the adequacy of current safety frameworks.

4.4 Biologic registries, traceability and future directionsBiologic registries are prospective, longitudinal observational studies on patients exposed to biologic drugs which collect data on outcomes such as therapeutic efficacy or adverse effects [177, 178]. They often span over more years than clinical trials with large sample sizes, thereby having potential to provide more of an insight to the long-term safety profiles of drugs. The British Society of Rheumatology led the way with the establishment of the British Society of Rheumatology Biologics Register for Rheumatoid Arthritis (BSRBR) in 2001, which has provided accurate monitoring of risks in conditions such as rheumatoid arthritis [179, 180]. However, there are currently no registries for biologics used in MS and in this respect, the neurology community has notably lagged behind other specialties. Clearly with the expanding use of biologics in the treatment of MS, having biologic registries can help identify the rare, but significant immunogenic adverse effects these drugs can cause.

Given biologics are often subjected to manufacturing changes and reformulation, it is crucial these products can be traced in the event of increased adverse outcomes to both the specific manufacturer and batches of the same product, to provide adequate information to investigate as to whether these changes contributed to increased adverse outcomes[181, 182]. Current challenges facing traceability systems in Europe is that they do not consistently record data such as batch numbers [181, 182] which could hinder any investigation into adverse effects caused by manufacturing changes. The introduction of standardised barcodes that encode manufacturer details and batch numbers may improve traceability[181, 182].

5 ConclusionsBiologic therapies offer unprecedented efficacy in the treatment of MS, but all carry significant risks and pose specific safety challenges. These risks are largely associated with the complex immunological effects of biologics. It is not clear that the current pharmacovigilance framework which underpins biologic safety is optimally configured to meet the complex safety issues associated with these drugs.

Compliance with Ethical Standards

FundingNo sources of funding were used to assist in the preparation of this review.

Conflicts of InterestKaty Murray has received financial support from Merck Serono, Biogen, Genzyme Sanofi, Roche and Novartis pharmaceuticals for scientific meeting attendance and consultation fees. David Hunt and Babak Soleimani have no conflicts of interest relevant to this review.

Figure 1 Timeline of FDA/EMA; approvals of biologic therapies for the treatment of multiple sclerosis. Approvals for recombinant interferon therapies were granted in the period 1995-2002, with further approvals for pegylated versions. Natalizumab was the first monoclonal antibody therapy approved for the treatment of multiple sclerosis, in 2005. Daclizumab has since been withdrawn.

202020152010200520001995

Interferon β1-b Interferon β1-a

Recombinant proteins

OcrelizumabDaclizumabNatalizumab Alemtuzumab

Monoclonal antibodies

Figure 2 Mechanism of action of monoclonal antibodies used to treat multiple sclerosis. See text for details. Daclizumab acts on CD25+ lymphocytes including NK cells. Rituximab (off-label) and Ocrelizumab are cell-depleting monoclonal antibodies which deplete B cells. Alemtuzumab causes lymphocyte depletion in cells which express CD52. Natalizumab inhibits transmigration of lymphocytes across the blood brain barrier through effects on integrin-adhesion molecule pairing.

Figure 3 Overview of immunological complications of biologic therapies for the treatment of multiple sclerosis (MS). Biologics can cause adverse effects through both direct or indirect immunogenic mechanisms. Direct mechanisms occur when a biologic drug results in increased or decreased immune function. Increased immune function results in secondary autoimmune diseases (e.g. Goodpasture’s syndrome secondary to alemtuzumab-induced anti-glomerular basement membrane antibodies), whilst decreased immune function results in opportunistic infection or cancers (e.g. PML secondary to natalizumab-induced focal CNS suppression). Indirect mechanisms occur through neutralising antibodies which are host antibodies developed against a biologic agent. These can alter the bioavailability of the drug, thus altering its therapeutic efficacy, or they can have pathogenic effects such as increasing risk of disease progression in MS.

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