IMMUNOGLOBULIN DRIVEN B CELL DISEASE

Technical Field

This invention relates to a compound and pharmaceutical compositions containing such compound for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease, a pathogenic immunoglobulin driven B cell disease with a T cell component or a pathogenic IgE driven B cell disease.

Background to the invention

The compound associated with this invention is known as clozapine i.e. the compound of the following structure:

Clozapine has a major active metabolite known as norclozapine (Guitton et al., 1999) which has the following structure:

Clozapine is known as a treatment for resistant schizophrenia. Schizophrenia is an enduring major psychiatric disorder affecting around 1% of the population. Apart from the debilitating psychiatric symptoms it has serious psychosocial consequences with an unemployment rate of 80-90% and a life expectancy reduced by 10-20 years. The rate of suicide among people with schizophrenia is much higher than in the general population and approximately 5% of those diagnosed with schizophrenia commit suicide.

Clozapine is an important therapeutic agent and is included on the WHO list of essential medicines.

It is a dibenzo-diazepine atypical antipsychotic, and since 1990 the only licensed therapy in the UK for the 30% of patients with treatment-resistant schizophrenia (TRS). It shows superior efficacy in reducing both positive and negative symptoms in schizophrenic patients and is effective in approximately 60% of previously treatment refractive patients with a significant reduction in suicide risk. The National Institute for Health and Clinical Excellence (NICE) guideline recommends adults with schizophrenia which has not responded adequately to treatment with at least 2 antipsychotic drugs (at least one of which should be a non-clozapine second generation antipsychotic) should be offered clozapine.

Clozapine is associated with serious adverse effects including seizures, intestinal obstruction, diabetes, thromboembolism, cardiomyopathy and sudden cardiac death. It can also cause agranulocytosis (cumulative incidence 0.8%); necessitating intensive centralised registry based monitoring systems to support its safe use. In the UK there are three electronic registries

(www.clozaril.co.uk. www.denzapine.co.uk and www.ztas.co.uk) one for each of the clozapine suppliers. Mandatory blood testing is required weekly for the first 18 weeks, then every two weeks from weeks 19-52 and thereafter monthly with a 'red flag' cut-off value for absolute neutrophil count (ANC) of less than 1500/pL for treatment interruption.

In 2015, the Federal Drug Administration (FDA) merged and replaced the six existing clozapine registries in the United States combining data from over 50,000 prescribers, 28,000 pharmacies and 90,000 patients records into a single shared registry for all clozapine products, the Clozapine Risk Evaluation and Mitigation Strategy (REMS) Program (www.clozapinerems.com). Changes were introduced lowering the absolute neutrophil count (ANC) threshold to interrupt clozapine treatment at less than 1000/pL in general, and at less than 500/pL in benign ethnic neutropenia (BEN).

Prescribers have greater flexibility to make patient-specific decisions about continuing or resuming treatment in patients who develop moderate to severe neutropenia, and so maximize patient benefit from access to clozapine.

Schizophrenia is associated with a 3.5 fold increased chance of early death compared to the general population. This is often due to physical illness, in particular chronic obstructive pulmonary disease (COPD) (Standardised Mortality Ratio (SMR) 9.9), influenza and pneumonia (SMR 7.0). Although clozapine reduces overall mortality in severe schizophrenia, there is a growing body of evidence linking clozapine with elevated rates of pneumonia-related admission and mortality. In an analysis of 33,024 patients with schizophrenia, the association between second generation antipsychotic medications and risk of pneumonia requiring hospitalization was highest for clozapine with an adjusted risk ratio of 3.18 with a further significant increase in risk associated with dual antipsychotic use (Kuo et al., 2013). Although quetiapine, olanzapine, zotepine, and risperidone were associated with a modestly increased risk, there was no clear dose-dependent relationship and the risk was not significant at time points beyond 30 days (Leung et al., 2017; Stoecker et al., 2017) .

In a 12 year study of patients taking clozapine, 104 patients had 248 hospital admissions during the study period. The predominant admission types were for treatment of either pulmonary (32.2%) or gastrointestinal (19.8%) illnesses. The commonest pulmonary diagnosis was pneumonia, (58% of pulmonary admissions) and these admissions were unrelated to boxed warnings (Leung et al., 2017).

In a further nested case control study clozapine was found to be the only antipsychotic with a clear dose-dependent risk for recurrent pneumonia, this risk increased on re-exposure to clozapine (Hung et al., 2016).

While these studies underscore the increased admissions or deaths from pneumonia and sepsis in patients taking clozapine over other antipsychotics, the focus on extreme outcomes (death and pneumonia) may underestimate the burden of less severe but more frequent infections such as sinusitis, skin, eye, ear or throat infections and community acquired and treated pneumonia.

Infection may represent an important additional factor in destabilizing schizophrenia control and clozapine levels.

Various mechanisms for the increase in pneumonia have been suggested, including aspiration, sialorrhoea and impairment of swallowing function with oesophageal dilatation, hypomotility and agranulocytosis. In addition, cigarette smoking is highly prevalent among patients with schizophrenia as a whole and represents an independent risk factor for pneumonia incidence and severity (Bello et al., 2014).

A small amount of research into the immunomodulatory properties of clozapine has been performed:

Hinze-Selch et al (Hinze-Selch et al., 1998) describes clozapine as an atypical antipsychotic agent with immunomodulatory properties. This paper reports during the initial 6 weeks of clozapine treatment, patients with schizophrenia showed significant increases in the serum concentrations of total IgG, but no significant effect was found on IgA or IgM concentrations or on the pattern of autoantibodies.

Jolles et al (Jolles et al., 2014) reports studies on the parameter "calculated globulin (CG)" as a screening test for antibody deficiency. Patients with a wide range of backgrounds were selected from thirteen laboratories across Wales. Of the patients with significant antibody deficiency (IgG <4g/L, reference range 6-16g/L), identified on CG screening from primary care, clozapine use was mentioned on the request form in 13% of the samples. However, antibody deficiency is not a listed side effect of clozapine in the British National Formulary (BNF), nor does antibody testing constitute part of current clozapine monitoring protocols.

A recent study by the same group (Ponsford et al., 2018b) follows up on this with a cross-sectional observational study to compare total immunoglobulin levels in patients prescribed clozapine or an alternative antipsychotic drug. They confirmed a significant reduction in immunoglobulin levels and increased proportion of patients using more than 5 courses of antibiotic annually in those on clozapine. The precise immune cellular correlates of this and causality were not investigated or feasible with the study design however. The study authors suggested that their findings, if confirmed, could hold implications for monitoring and risk mitigation approaches in patients with schizophrenia prescribed clozapine (Ponsford et al., 2018b).

Another study by Lozano et al. (Lozano et al., 2016) reported an overall decrease of mean plasma levels of IgM in the study group (which consisted of psychiatric outpatients who took clozapine for at least five years) compared to the control group, and also reported that no differences were found between the groups with respect to IgA, IgG, absolute neutrophil count and white blood cell count.

Consequently, given these mixed results that have been reported, the immunomodulatory properties of clozapine and its specific effect on immunoglobulin levels, particularly with respect to cellular substrate and immunophenotypic impact beyond total immunoglobulin levels are neither clear nor understood in the art.

Compounds for therapeutic use are available in suitable forms in adequate quantities. However, such compounds may be modified to improve pharmacodynamics and pharmacokinetics, and decrease immunogenicity. One such method commonly used for to modify compounds is by covalent attachment of water soluble polymers. Polyethylene glycol ("PEG") is one such chemical moiety which has been used in the PEGylation of therapeutic compound products. The US FDA has approved PEG for use as a vehicle or base in foods, cosmetics and pharmaceuticals, including injectable, topical, rectal and nasal formulations. Molecules coupled to PEG become non-toxic, non- immunogenic, soluble in water and many organic solvents, and surfaces modified by PEG attachment become hydrophilic and protein rejecting. Pegylated derivatives of clozapine and norclozapine have been described in GB1562874. Pathogenic immunoglobulin driven diseases including pathogenic immunoglobulin (particularly IgG and IgA) driven B cell diseases, pathogenic immunoglobulin driven B cell diseases with a T cell component and pathogenic IgE driven B cell diseases result from secretion of autoantibodies by antibody secreting cells ("ASCs", collectively plasmablasts and plasma cells, these being types of mature B cell), and /or B cell-T cell cross-talk/interaction. These antibodies target a variety of self antigens which have been characterised in many of these conditions. There is rarely an increase in overall immunoglobulins as the pathological process is driven by the secretion of specific immunoglobulins which constitute a small percentage of the total immunoglobulins. Secretion of IgG, IgA or IgE antibodies are from ASCs, and ASCs are generated secondary to the differentiation of class-switched and unswitched memory B cells, these being further types of mature B cell, with differentiation occurring both within and without germinal centres. Various lines of evidence suggest this is a highly-dynamic process, with ongoing differentiation occurring almost

constantly. The T cell component that contributes towards the pathology of the diseases arises in part because B cells act as professional antigen-presenting cells for T cells (their importance is increased also due to their sheer numbers). B cells secrete significant amounts of cytokines that impact T cells and B-T cell interaction is involved in response to T dependent protein antigens and class switching. In addition, T cells themselves provide specific 'help' to B cells, for example in the setting of the germinal centre reaction whereby activated CD4 T follicular helper cells interact with activated B cells , with the interaction promoting affinity maturation, class switching and differentiation of B cells into memory and long-lived plasma cells (Shulman et al., 2013; Valentine et al., 2018). T cell-B cell interdependence and dynamic collaboration (mediated by pathways and costimulatory receptors including CD40/CD40L, CD28/CTLA-4, 0X40, ICOS, signal lymphocyte activation molecule (SLAM) family receptors and cytokines such as IL-12, IL-4 and IL-21) is critical for germinal centre formation and B cell differentiation into functionally distinct memory cells (Koike et al., 2019; Petersone et al., 2018; Shulman et al., 2014). T cell-B cell interaction also directs B cell entry towards commitment to a plasma cell or recycling germinal centre cell fate and is, accordingly, vital for humoral immunity (Ise et al., 2018). T cells will therefore contribute in a number of ways to the activity and the maturation of B cells.

Class-switched memory B cells are mature B cells that have replaced their primary encoded membrane receptor IgM by IgG, IgA or IgE (the latter also via sequential switching from IgGl in addition to direct class-switching from IgM) (He et al., 2017) in response to repeated antigen recognition. This class-switching process is a key feature of normal humoral immunological memory, both 'constitutive' through the secretion of pre-existing protective antibodies by long-lived plasma cells, and 'reactive' reflecting re-exposure to antigen and reactivation of memory B cells to either differentiate into plasma cells to produce antibodies, or to germinal centre B cells to enable further diversification and affinity maturation of the antibody response. Early in the immune response, plasma cells derive from unswitched activated B cells and secrete IgM. Later in the immune response, plasma cells originate from activated B cells participating in the germinal centre (areas forming in secondary lymphoid follicular tissue in response to antigenic challenge) which have undergone class switching (retaining antigen specificity but exchanging immunoglobulin isotype) and B cell receptor (BCR) diversification through immunoglobulin somatic hypermutation. This maturation process enables the generation of BCRs with high affinity to antigen and production of different immunoglobulin isotypes (i.e. exchanging the originally expressed IgM and IgD to IgG, IgA or IgE isotypes) (Budeus et al., 2015; Kracker and Durandy, 2011).

Class switch recombination (CSR) following the germinal centre reaction in secondary lymphoid organs provides antigen-primed/experienced autoreactive memory B cells and a core pathway for development and/or maintenance of autoimmunity. Post-germinal centre B cells class-switched to IgG, IgA or IgE in the periphery can enter other anatomic compartments, such as the central nervous system, to undergo further affinity maturation (e.g. in tertiary lymphoid structures in multiple sclerosis) and contribute to immune pathology (Palanichamy et al., 2014). CSR can occur locally within tissue in pathology, such as within ectopic lymphoid structures in chronically inflamed tissue such as rheumatoid arthritis synovium (Alsaleh et al., 2011; Humby et al., 2009).

A significant proportion of bone marrow plasma cells are lgA ⁺ (~40%) with lgA ⁺ plasma cells further constituting the majority in serum (~80%) (Mei et al., 2009) consistent with a substantial contribution of lgA ⁺ plasma cells to the bone marrow population of long-lived cells. The intestinal mucosa is the primary inductive site for lgA ⁺ plasma cells, mainly through gut-associated lymphoid tissue (GALT, comprising Peyer's patches and isolated lymphoid follicles) (Craig and Cebra, 1971), together with mesenteric lymph nodes and, potentially, the intestinal lamina propria itself, with class-switch recombination towards IgA achieved through both T cell-independent (pre-germinal centre formation) (Bergqvist et al., 2010; Casola et al., 2004) and T cell-dependent mechanisms (Pabst, 2012). Notably, lgA ⁺ and other plasma cells (in addition to plasmablasts) are increasingly understood to exert important effector immune functions beyond the production of

immunoglobulin, including generation of cytokines (Shen and Fillatreau, 2015) and

immunoregulators such as tumour-necrosis factor-a (TNF-a), inducible nitric oxide synthase (iNOS) (Fritz et al., 2011), IL-10 (Matsumoto et al., 2014; Rojas et al., 2019), IL-35 (Shen et al., 2014), IL-17a (Bermejo et al., 2013) and ISG15 (Care et al., 2016). In the case of IgE memory responses, these have been shown to arise from a distinct pathway involving a transient germinal centre phase, affinity maturation and class-switching via an IgG memory B cell intermediate and rapid development towards a plasma cell fate (He et al., 2017;

Xiong et al., 2012). In humans, the majority of IgE cells are plasma cells (or plasmablasts) rather than memory cells (Saunders et al., 2017) likely due to a transient apoptotic germinal centre phase, paucity of IgE memory B cells and propensity for lgE+ B cell differentiation to plasma cells, the source of allergen-specific IgE central to allergic responses (Ramadani et al., 2017; Saunders et al., 2019).

Plasmablasts, representing short-lived rapidly cycling antibody-secreting cells of the B cell lineage with migratory capacity, are also precursors to long-lived (post-mitotic) plasma cells, including those which home in to the bone marrow niche (Nutt et al., 2015). In addition to being precursors of autoreactive long-lived plasma cells, plasmablasts are an important potential therapeutic target themselves through their ability to produce pathogenic immunoglobulin/ autoantibody (Hoyer et al., 2004), particularly IgG but also IgM and IgE, described in several disease contexts such as neuromyelitis optica (Chihara et al., 2013; Chihara et al., 2011), idiopathic pulmonary arterial hypertension, lgG4-related disease (Wallace et al., 2015), multiple sclerosis (Rivas et al., 2017) and transverse myelitis (Ligocki et al., 2013), rheumatoid arthritis (Owczarczyk et al., 2011), systemic lupus erythematosus (SLE) (Banchereau et al., 2016) and IgE-mediated allergic disorders (e.g.

asthma, food allergy and atopic dermatitis) (Heeringa et al., 2018). In addition to their direct antibody secreting function, circulating plasmablasts also exert activity to potentiate germinal centre-derived immune responses and thereby antibody production via a feed-forward mechanism involving ll-6-induced promotion of T follicular helper cell (Tfh) differentiation and expansion (Chavele et al., 2015).

Long-lived plasma cells, whose primary residency niche is in bone marrow (Benner et al., 1981), are thought to be the major source of stable autoantibody production in (both physiologic) and pathogenic states and are resistant to glucocorticoids, conventional immunosuppressive and B cell depleting therapies (Hiepe et al., 2011). Substantiating the critical importance of this B cell population to long-term antibody production, site-specific survival of bone marrow-derived plasma cells with durable (up to 10 years post-immunisation) antibody responses to prior antigens has been demonstrated in non-human primates despite sustained memory B cell depletion (Hammarlund et al., 2017). Given the key role played by autoreactive long-lived plasma cells in the maintenance of autoimmunity (Mumtaz et al., 2012) - and the substantial refractoriness of the autoreactive memory formed by these cells to conventional immunosuppressive agents such as anti-TNF or B cell depleting biologies (Hiepe et al., 2011) - a specific effect to deplete bone marrow long-lived plasma cells has, via an impact on long-lived plasma cell (autoreactive) memory, substantial therapeutic potential in pathogenic immunoglobulin driven B cell disease to eliminate inflammation and achieve remission.

CD19(+) B cells and CD19(-) B plasma cells are drivers of pathogenic immunoglobulin driven B cell diseases, pathogenic immunoglobulin driven B cell diseases with a T cell component and pathogenic IgE driven B cell diseases. In particular, pathogenic IgG and IgA driven B cell diseases, pathogenic immunoglobulin driven B cell diseases with a T cell component and pathogenic IgE driven B cell diseases represent a substantial proportion of all autoimmune and inflammatory diseases. The most prominent, but not the sole mechanism through which pathogenic immunoglobulin driven B cells cause disease, is through auto-antibody production. Established pathogenic IgG immunoglobulin diseases include Pemphigus and Pemphigoid. Pemphigus, which has been designated to be an orphan disease, is an autoimmune interepithelial blistering disease characterised by loss of normal cell-cell adhesion (acantholysis). The antibodies involved are against desmoglein 3. If left untreated, it can be fatal, usually from overwhelming opportunistic infection due to loss of skin barrier function and from electrolyte loss. Pemphigoid is characterised by the formation of blister at the space between the epidermis and dermis skin layers. The antibodies involved are against dystonin and/or type XVII collagen.

Pathogenic immunoglobulin driven B cell diseases, pathogenic immunoglobulin driven B cell diseases with a T cell component and pathogenic IgE driven B cells diseases are poorly treated and as a result they have substantial mortality and morbidity rates, even for the more "benign" diseases. Certain current advanced therapies are directed at mature B cells. For example, belimumab is a human monoclonal antibody that inhibits B cell activating factor. Atacicept is a recombinant fusion protein that also inhibits B cell activating factor. However, memory B cells may be resistant to therapies such as belimumab or atacicept which target survival signals such as B cell activation factor (Stohl et al., 2012). The importance of memory B cells in the pathogenesis of autoimmune disorders was also demonstrated by the lack of efficacy of atacicept in treating rheumatoid arthritis and multiple sclerosis (Kappos et al., 2014; Richez et al., 2014). Plasmapheresis and immunoadsorption involve the removal of disease-causing autoantibodies from the patient's bloodstream. However, these treatments have limited efficacy or are complex and costly to deliver. CAR-T methods directed at CD19(+) B cells leaves CD19(-) B plasma cells intact, which makes it ineffective.

Rituximab is a drug that is currently used to treat some pathogenic IgG driven B cell diseases. It targets B cells that express CD20. However, CD20 is only expressed on a limited subset of B cells. It also does not target plasma cells. This limited expression of CD20 and lack of effect on plasma cells explains the limited efficacy of rituximab in a variety of diseases, both benign and malignant, despite being definitively of B cell origin. Rituximab does not appear to have any effect on IgA-secreting plasmablasts/plasma cells, and consequently the associated IgA driven B cell diseases (Yong et al 2015). The effect of rituximab on IgE levels is modest and no sustained clinical benefit has been observed (van Vollenhoven et al., 2013). Omalizumab (anti-lgE antibody) is presently indicated for the treatment of inadequately controlled severe allergic persistent asthma. It is, however, an expensive medicine.

Thus, there is a major unmet medical need for new treatments against pathogenic immunoglobulin driven B cell diseases, pathogenic immunoglobulin driven B cell diseases with a T cell component and pathogenic IgE driven B cell diseases.

Summary of the invention

It has been found by the inventors that clozapine has a potential important therapeutic effect as it significantly reduces class switched memory B cells ("CSMB"), a type of mature B cell.

Further, it has been found by the present inventors that clozapine treatment in humans is associated with a significant reduction in immunoglobulin levels, for example IgG and IgM subtypes, and impaired responses to vaccination with T-independent unconjugated pneumococcal polysaccharide antigens and T-dependent protein antigens (e.g. Hib) confirming both a quantitative and qualitative impact on B cell antibody production. In addition, there is a significant reduction in levels of class switched memory B cells (CSMB) and an observed reduction in levels of plasmablasts, both types of mature B cell. CSMB are antigen activated mature B cells that no longer express IgM or IgD and instead express the immunoglobulins IgG, IgA or IgE. They are significant antibody producers.

Plasmablasts are also mature B cells which are significant antibody producers, being at a later stage of maturity than CSMBs. A reduction in levels of CSMB indicates that clozapine has an effect on the pathways involved in B cell maturation on the way to the production of mature plasma cells. B cells are also professional antigen presenting cells and cytokine producers and have a role in CD4 T cell priming. The inventors' new data also demonstrates an effect of the drug in reducing total IgG, IgA and IgM levels after administration. With the lack of effect on other B cells, shown by the lack of depletion of other sub-types and total B cell numbers but with a particular reduction in CSMBs and plasmablasts, this observation strongly supports a functional effect on CSMBs and plasmablasts which are central to long lived production of pathogenic antibodies in pathogenic immunoglobulin driven B cell disease with a T cell component.

Impact on class-switched memory B cells and antibody production Reduction in CSMBs by clozapine will consequently reduce the numbers of ASCs, and hence the secretion of specific immunoglobulins including the pathogenic immunoglobulins. Clozapine was also observed to cause a reduction in levels of plasmablasts, another type of mature B cell. This functional effect on persistent and long-lived adaptive B cell and plasma cell function may ameliorate the diseases driven by the persistent generation of pathogenic immunoglobulins that drives the pathology of pathogenic immunoglobulin driven B cell diseases, pathogenic

immunoglobulin driven B cell diseases with a T cell component and pathogenic IgE driven B cell diseases. The inventors' new data demonstrates a very significant effect on the number of circulating class switched memory B cells, a substantial effect on the number of plasmablasts and importantly, through the lack of recall response to common vaccines, an effect on the function of the class switched memory B cells and plasmablasts resulting in specific reduction of antibodies targeting a previously exposed antigen. The inventors' new data also demonstrates an effect of the drug in reducing total immunoglobulin levels after administration. With the lack of effect on other B cells, shown by the lack of depletion of other sub-types and total B cell numbers but with a particular reduction in CSMBs and plasmablasts, this observation strongly supports a functional effect on CSMBs and plasmablasts which are central to long lived production of pathogenic antibodies in pathogenic immunoglobulin (particularly IgG and IgA) driven B cell diseases, pathogenic

immunoglobulin driven B cell diseases with a T cell component and pathogenic IgE driven B cell diseases.

The inventors' finding of a marked reduction in class-switched memory B cells in patients treated with clozapine indicates a robust impact on the process of immunoglobulin class switching. This has particular therapeutic relevance in pathogenic immunoglobulin driven B cell diseases in which class switch recombination (CSR) following the germinal centre reaction in secondary lymphoid organs provides antigen-primed/experienced autoreactive memory B cells and a core pathway for development and/or maintenance of autoimmunity. Further, this also has particular therapeutic relevance since the B lymphoid kinase haplotypes associated with B cell-driven autoimmune disorders exhibit an expansion of class-switched memory B cells and disease models of intrinsic B cell hyperactivity are associated with spontaneous CSR as associated with high titres of IgG

autoantibodies The effect of clozapine to both impact on CSR and lower IgG is of especial therapeutic potential in the setting of pathogenic immunoglobulin-driven B cell diseases where an impact on both the autoimmune memory repertoire and pathogenic immunoglobulin is desirable.

Impact on IgA The inventors have identified a significantly reduced circulating total IgA in patients treated with clozapine (leftward shift in immunoglobulin distribution) which notably demonstrated

disproportionate lowering of IgA compared to that found with IgG and IgM. Substantiating the functional impact of this, the inventors have also identified a highly significant reduction in pneumococcal-specific IgA in patients treated with clozapine compared to clozapine-naive patients taking other antipsychotics. Recapitulating this in a model mammalian system, the inventors demonstrate that dosing of wild type mice with clozapine results in a significant reduction in circulating IgA compared to control or haloperidol treatment. While present at a relatively lower concentration in plasma compared to other immunoglobulin isotypes, IgA forms the great majority of all mammalian immunoglobulin, with ~3 g/day produced in human (Macpherson et al., 2012).

The inventors' finding of a significant reduction in total IgA in response to clozapine treatment reflects an important effect of clozapine on the function of lgA ⁺ plasma cells. The generation of such cells occurs in both bone marrow and intestinal mucosae.

The inventors' identification of a significant impact of clozapine on plasma cell populations indicates the clear potential to modulate the diverse antibody-independent effector functions of B cells relevant to (auto)immune-mediated disease also.

Impact on plasmablast antibody-secreting cells

The inventors have found that clozapine exerts a profound effect on reducing levels of circulating plasmablasts in patients. Accordingly, the inventors' observation of a profound impact of clozapine use on circulating plasmablast number highlights the potential for clozapine to modulate pathogenic immunoglobulin-driven B cell disease through both effects on circulating plasmablast secretion of pathogenic immunoglobulin (Rivas et al., 2017; Stathopoulos et al., 2017) as well as interference with the potent function of plasmablasts to promote Tfh function.

Impact on long-lived plasma cells

Using a wild type murine model, the inventors have found that regular clozapine administration in mice significantly reduces the proportion of long-lived plasma cells in bone marrow, an effect not seen with use of a comparator antipsychotic agent (haloperidol). Notably, human bone marrow resident long-lived PCs are long-regarded as the primary source of circulating IgG in human, thus providing a clear substrate for the inventors' observation of reduction in IgG in patients treated with clozapine. The inventors' observation of a specific effect of clozapine to deplete bone marrow long- lived plasma cells has, via an impact on long-lived plasma cell (autoreactive) memory, substantial therapeutic potential in pathogenic immunoglobulin driven B cell disease to eliminate inflammation and achieve remission.

The inventors' identification of a significant impact of clozapine on plasma cell populations indicates the clear potential to modulate the diverse antibody-independent effector functions of B cells relevant to (auto)immune-mediated disease also.

Impact on B cell precursors in bone marrow and splenic immature/transitional cells

The inventors identify a clear impact of clozapine on bone marrow B cell precursors after dosing of wild type mice. Specifically, an increase in the proportion of pre-pro B cells, in conjunction with a reduction in pre-B cells, proliferating pre-B cells and immature B cells in bone marrow. Together, these findings suggest a specific impact of clozapine on early B cell development, with a partial arrest between the pre-pro-B cell and pre-B cell stages in the absence of specific immunological challenge. The inventors have discerned an impact of clozapine to reduce the proportion of splenic T1 cells in wild type mice. Mirroring the murine findings, the inventors' interim findings from an ongoing observational study of patients on clozapine reveal a significant reduction in circulating transitional B cells. The human circulating transitional B cell subpopulation exhibits a phenotype most similar to murine T1 B cells and is expanded in patients with autoimmune disease.

Accordingly, the inventors' observation of an impact of clozapine to reduce the proportions of bone marrow B cell progenitors and immature (Tl) splenic B cells provides additional anatomic compartmental origins beyond germinal centres for their finding of a reduction in circulating class- switched memory B cells and immunoglobulin in patients treated with clozapine. The therapeutic potential of this is further underlined by the consideration that the majority of antibodies expressed by early immature B cells are self-reactive (Wardemann et al., 2003).

Lack of direct B cell toxicity in vitro

The inventors' new data using an in vitro B cell differentiation system to assess the specific impact of clozapine, its metabolite (N-desmethylclozapine; norclozapine) and a comparator antipsychotic control drug (haloperidol) further demonstrate: no direct toxicity effect of clozapine or its metabolite on differentiating B cells, no consistent effect on the ability of differentiated ASCs to secrete antibody and no consistent inhibitory effect on functional or phenotypic maturation of activated B cells to an early PC state in the context of an established in vitro assay.

Limited to the context of these in vitro experiments, these data suggest that clozapine is unlikely to be acting in a direct toxic manner on plasma cells or their precursors (e.g. via a cell intrinsic effect) to induce the effects observed on immunoglobulin levels. The observations suggest that clozapine's effect on B cells is more nuanced than existing B cell targeting therapies used for autoimmune disease which result in substantial depletion of multiple B cell subpopulations (e.g. rituximab and other anti-CD20 biosimilars) whose efficacy is mediated via direct effects on B cells such as signalling induced apoptosis, complement-mediated cytotoxicity or antibody-dependent cellular cytotoxicity.

Such a lack of apparent substantial direct toxicity by clozapine has a number of potential therapeutic advantages for clozapine, including reduced risk of generalised immunosuppression associated with indiscriminate B cell depletion (including elimination of protective B cells), and the potential to avoid maladaptive alterations observed with use of conventional B cell depleting therapies.

Efficacy in collagen-induced arthritis (CIA) mouse model, relevance of CIA as a model of pathogenic immunoglobulin (IgA, IgG, IgM and lgE)-driven B cell disease and pathogenic immunoglobulin driven B cell disease with a T cell component, and importance of B cell-T cell interactions in autoimmunity

CIA is a well-established experimental model of autoimmune disease that results from

immunisation of genetically susceptible strains of rodents and non-human primates with type II collagen (CM) (Brand et al., 2004) - a major protein component of cartilage - emulsified in complete Freund's adjuvant. This results in an autoimmune response accompanied by a severe polyarticular arthritis, typically 18-28 days post-immunisation and monophasic, resolving after ~60 days in mice (Bessis et al., 2017; Brand et al., 2007). The pathology of the CIA model resembles that of rheumatoid arthritis, including synovitis, synovial hyperplasia/pannus formation, cartilage degradation, bony erosions and joint ankylosis (Williams, 2012).

The immunopathogenesis of CIA is dependent on B cell-specific responses with generation of pathogenic autoantibodies to CM, in addition to involving T cell-specific responses to CM, FcyR (i.e. Fc receptors for IgG) and complement. The critical role of B cells in the development of CIA is substantiated by the complete prevention of development of CIA in mice deficient for B cells (IgM deleted), notwithstanding an intact anti-CM T cell response (Svensson et al., 1998). Moreover, the development of CIA has been shown to be absolutely dependent on germinal centre formation by B cells, with anti-CII immunoglobulin responses themselves largely dependent on normal germinal centre formation (Dahdah et al., 2018; Endo et al., 2015). B cells have also been implicated in other aspects of CIA pathology, including bone erosion through inhibition of osteoblasts (Sun et al.,

2018b). As a corollary, B cell depletion using anti-CD20 monoclonal antibodies prior to CM immunisation delays onset and severity of CIA, in conjunction with delayed autoantibody production (Yanaba et al., 2007). In this model, B cell recovery was sufficient to result in pathogenic immunoglobulin production after collagen-immunisation and associated development of disease. The fundamental role played by collagen-specific IgG autoantibodies in the pathogenesis of CIA are highlighted by the observations that passive transfer of anti-CII serum or polyclonal IgG

immunoglobulin to unimmunised animals results in arthritis (Stuart and Dixon, 1983), whilst lack of the FcyR chain near completely abrogates development of CIA in mice (Kleinau et al., 2000). In addition, introduction of pathogenic antibodies (i.e. collagen antibody-induced arthritis, CAIA) into germinal centre-deficient mice results in arthritis, demonstrating the ability of pathogenic antibody to largely circumvent the requirement for the germinal centre reaction (Dahdah et al., 2018).

Moreover, even mice lacking adaptive immunity (i.e. B and T cells), are susceptible to induction of CIA (Nandakumar et al., 2004).

Dynamic interactions between B cells and T cells are critical to an adaptive immune response and contribute to pathogenic immunoglobulin production in disease. Exemplifying this is the germinal cell reaction through which high affinity long-lived memory B cells and plasma cells are generated. B cell differentiation to these distal mature cell types requires both B cell activation and multi-stage selection/survival signals provided by mature T follicular helper cells to germinal centre B cells delivered focally via immunological synapses enabling kinetic, temporal and spatial segregation of multiple (bidirectional) signalling/co-stimulatory molecules and cytokines (Allen et al., 2007), including CD40L-CD40 (Foy et al., 1994), IL-21 (the most potent cytokine promoting plasma cell differentiation) (Ettinger et al., 2005; Kuchen et al., 2007; Zotos et al., 2010), PD-1/PD-L1 (Dorfman et al., 2006; Good-Jacobson et al., 2010), ICOS-ICOSL (Choi et al., 2011; Liu et al., 2015; Xu et al., 2013), SLAM (signaling lymphocyte activation molecule) family receptors (Cannons et al., 2010) required for sustained B ceILT cell adhesion and others. This process of 'entanglement' is critical to selective delivery of helper signals to high affinity, non-autoreactive B cell clones to select for plasma cell differentiation. Underlining the importance of T follicular helper cells (T _F H) in the generation of B cell memory, T _F H cells and their PI3K6 activity are the primary limiting factor in germinal centre development (Rolf et al., 2010). T _F H cells also secrete class switch factors required to instruct class switch recombination of B cells (Crotty, 2011), including IL-4 for IgGl (Reinhardt et al., 2009) and IgE, IL-21 for lgG3, IgA and IgE (Avery et al., 2008; Pene et al., 2004). Notably, the process of B cell-T cell interaction in lymphoid tissue is not restricted to germinal centre T _FH -germinal centre B cell interactions, but also includes (Tangye et al., 2015): extrafollicular T cell help to plasmablasts via IL- 21 and Bcl-6 (Lee et al., 2011) supported by stromal cell-derived APRIL (Zhang et al., 2018) , Tm-non- cognate B cell interactions in the follicular mantle and cognate interactions at the T-B border.

Notably these interactions are not solely unidirectional; thus, circulating plasmablasts can reciprocally modulate T _F H cells and promote the T _F H differentiation programme via secretion of IL-6 (Chavele et al., 2015). This positive feedback loop and the earlier observations underline the interdependence of B cell and T cell responses to physiological and pathological immunoglobulin production and the genesis/perpetuation of autoimmunity.

Cognate interactions between B cells and T cells are recognised as critical to the induction of CIA. Accordingly, blocking the interaction of CD40 ligand (gp39) expressed on the surface of CD4 ⁺ T (helper) cells with CD40 on the surface B cells using monoclonal anti-CD40-L antibodies is sufficient to completely prevent the development of CIA in mice with associated reduction in pathogenic anti- Cll antibodies (Durie et al., 1993). Similarly, T cell-B cell ICOS signalling has been shown to be necessary for the induction and maintenance of CIA in mice (Panneton et al., 2018); as a corollary, inhibition of the ICOS/ICOS-L interaction reduces disease severity and progression in mice (O'Dwyer et al., 2018). Further, IL-21 knockout mice are resistant to the development of CIA and exhibit lower IgG anti-CII antibodies, with 11-21 signalling in B cells shown to be responsible for CIA development (Sakuraba et al., 2016).

An additional T cell population shown to play a role in (suppression of) humoral immunity are Foxp3 ⁺ regulatory T cells (Tregs). Underlining the importance of Tregs, their depletion using anti-CD25 or diphtheria toxin results in potent induction of autoantibodies, enhanced T _F H cell and germinal centre responses and histological evidence of autoimmunity (Leonardo et al., 2012; Sakaguchi et al., 1995). Specifically, within secondary lymphoid tissue T follicular regulatory cells residing at the T cell zone-B cell follicle border and B cell follicle (Sayin et al., 2018) act to inhibit antibody production through multiple interactions with B cells and T _F H cells, with mechanisms proposed (Wing et al., 2018) including: direct suppression of follicular b cells, prevention of T _F H cell germinal centre entry and inhibition of B cell differentiation in the germinal centre itself. Regulatory T cells therefore modulate the differentiation of antibody secreting cells via germinal centres through their co-option of the T _F H differentiation pathway (Chung et al., 2011; Linterman et al., 2011). Underlining the importance of Treg cells in the pathogenesis of CIA, adoptive transfer of antigen-specific Treg cells inhibits the progression of CIA (Sun et al., 2018a).

The present inventors have found that clozapine leads to a significant reduction in the proportion of B cells in lymph nodes of mice immunised with heterologous type II collagen. Concordant findings of smaller magnitude were evident in spleen. A similar reduction was observed when dosing healthy wild type mice with clozapine without predilection for a particular major B cell subset, suggesting an influence of clozapine to reduce major secondary lymphoid tissue B cell subsets.

The inventors' data also shows a highly significant ability of clozapine to reduce the proportion of germinal centre B cells, together with a very significant dose-dependent reduction in their levels of activation, as judged by their expression of the GL7 activation antigen/epitope. Notably GL7 ^hl B cells show greater specific and total antibody production in addition to greater antigen presenting capacity. Accordingly, the inventors' finding suggests that clozapine has effects on both the abundance of germinal centre B cells as well as their functionality, with both effects converging to inhibit effective germinal centre function and/or formation.

In addition, the inventors have identified an additional effect of clozapine on the other major cell type critical for germinal centre formation and function, namely T follicular helper cells (T _F H). They find that clozapine substantially reduces expression of key T _F H markers, PD-1 (programmed cell death-1) and CXCR5 without a perturbation in the proportion of T _F H cells in secondary lymphoid tissue. T _F H cells express PD-1 at high levels (and upregulate expression soon after antigen stimulation) where it serves to critically regulate T _F H position and function in the germinal centre. Specifically, when engaged by surrounding follicular B cells which constitutively express the PD-1 ligand (PD-L1), PD-1 acts to inhibit T cell recruitment into the follicle thereby concentrating T _F H cells into the germinal centre itself. This is critical for T _F H cells to undertake their proper role to support germinal centre B cells. PD-1 is also required for optimal IL-21 production by T _F H cells. As a corollary PD-1 deficient mice have fewer long-lived plasma cells, in part due to greater germinal centre cell death. Within the germinal centre the PD-1/PD-L1 interaction also serves to optimise B cell competition and affinity maturation.

Concordantly, the inventors also observe a highly significant impact of clozapine to reduce expression of CXCR5 on T _F H cells. CXCR5 is regarded as a defining marker for T _F H cells and is required for T cell follicular homing. Notably T cells deficient in CXCR5, while able to access the follicular germinal centre, are inefficient at supporting GC responses.

Thus, the inventors' findings indicate that clozapine exerts an inhibitory influence on T _F H

functionality and germinal centre formation, at least in part through altered expression of PD-1 and CXCR5. The findings indicate that clozapine reduces the ability of T _F H cells to concentrate within the germinal centre to provide B cell help to support differentiation of antigen specific B cells into plasma cells and memory cells and lowers the efficiency thereof, thereby exerting a potent inhibitory influence on antibody dependent immune responses. Overall, the findings of lowered cell surface expression of CXCR5 and PD-1 are suggestive of an impairment in T _F H cells to differentiate into true mature germinal centre T _F H cells.

In addition, the inventors show that clozapine increases the proportion of Foxp3 ⁺ regulatory T cells, an immune suppressive T cell population, (Tregs) in secondary lymphoid tissue (draining lymph node and spleen) in addition to upregulating expression of CD25 on Foxp3 ⁺ Tregs. In the context of lymphoid follicles, Foxp3 ⁺ T follicular regulatory cells (Tfr) regulate the germinal centre reaction, serving to limit germinal centre B cell and T _F H numbers, and inhibit antibody affinity maturation, plasma cell differentiation and antigen-specific immunoglobulin secretion. Accordingly, the inventors' findings suggest that clozapine is likely to act in part through Treg-B cell interaction (in addition to provision of T cell help to B cells) to dampen humoral immune responses.

IgE memory B cells and IgE plasma cells have also been shown to develop via a germinal centre pathway (Talay et al., 2012). Notably IgE switch memory B cells are the main source of cellular IgE memory (Talay et al., 2012). Moreover the ontogeny of lgE ⁺ B cells and plasma cells follows similar phenotypic stages to that for IgG(l), including lgE ⁺ germinal centre-like B cells, lgE ⁺ plasmablasts and lgE ⁺ plasma cells occurring via a sequential switching process from IgG (Ramadani et al., 2017). Notably the intrinsic maturation state of B cells determines their capacity to undergo class switching to IgE, accordingly the highest proportion of lgE ⁺ cells derive from germinal centre B cells (Ramadani et al., 2017). Furthermore, isotype switching depends on the number of cell divisions and is greater for IgE than IgG (Tangye et al., 2002), consistent with the fact that IgE responses generally require more prolonged antigenic stimulation (Hasbold et al., 1998). Accordingly, the inventors' findings of a specific impact of clozapine on class switching, germinal centre formation and long-lived plasma cells are expected to impact substantially on the ability to mount and sustain an IgE-mediated immunoglobulin response in pathogenic IgE driven B cell diseases. Indeed, the greater number of B cell divisions and requirement for germinal centre B cells to efficiently generate lgE ⁺ suggests that these disorders may be particularly susceptible to the effects of clozapine.

Accordingly, the inventors have employed the CIA model as a highly clinically relevant experimental system in which B cell-derived pathogenic immunoglobulin made in response to a sample specific antigen or antigen following B cell-T cell interaction (including in draining lymph node germinal centres) (Dahdah et al., 2018) drives autoimmune pathology to explore the potential efficacy of clozapine and its associated cellular mechanisms. The inventors demonstrate that clozapine delays the onset and reduces the incidence of CIA in mice, an effect most apparent when dosed just after CM immunisation. Furthermore, the inventors' data indicates that clozapine reduces the severity of CIA, judged by number of affected paws and clinical severity score. The inventors identify important effects of clozapine on key cell types implicated in the pathogenesis of CIA, including a reduction in the proportion of splenic plasma cells and highly significant reduction in germinal centre B cells in local draining lymph node. Moreover, the inventors' findings demonstrate reduced markers of functional activity for antibody production and antigen presentation on lymph node germinal centre B cells in response to clozapine in CM immunised mice. Measured at a single time point, they also observe a significant reduction in anti-collagen IgGl antibody levels. Together, the inventors' findings in the CIA model point to a specific ability of clozapine to favourably impact upon pathogenic immunoglobulin B cell-driven pathology and thereby B cell mediated disorders in which autoantibody formation is a key component. Thus, the present invention provides a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease, a pathogenic immunoglobulin driven B cell disease with a T cell component or a pathogenic IgE driven B cell disease in a subject, in particular, wherein said compound causes mature B cells to be inhibited in said subject.

Effect of clozapine and norclozapine in healthy volunteers following primary vaccination with Typhim Vi

The inventors demonstrated that short term, low dose administration of clozapine in healthy volunteers who received primary immunisation with Typhim Vi was associated with a reduction in immunoglobulin level, in particular IgM and IgG, together with a rapid reduction in multiple circulating B cell subsets. Complete elimination of pre-switched (IgM-only) memory B cells was observed together with a negative correlation between plasma clozapine levels and multiple B cell subsets. The inventors finding suggests that clozapine's specific effects on multiple memory B cell subtypes may be of therapeutic utility in autoimmune and other disorders driven by dysregulation in the immune compartment, particularly with regards to autoreactive B cells and/or germinal centres.

Effect of clozapine and norclozapine in a Keyhole Limpet Haemocyanin (KLH) T cell-dependent antibody response (TDAR) model

The inventors have indicated that clozapine reduces both primary (IgM-dominated and early IgG response) and secondary humoral responses to immunisation with the KLH models. Specifically, the inventors' demonstrated that clozapine (i) induces a significant reduction in spleen weight of KLH- immunised mice; (ii) exerts a significant reduction in the acute IgM response to primary

immunisation with KLH-TNP; (iii) exerts potent suppressive effects on primary IgG response to KLH immunisation; (iv) reduces primary IgG antibody response; (v) suppresses the secondary antibody response to KLH consistent with a strong ability to dampen B cell memory responses; and (vi) suppresses secondary IgG antibody response.

In vitro B cell profiling of clozapine and norclozapine using human donor B cells

The inventors evaluated the potential for clozapine and norclozapine to exert direct effects on fundamental aspects of B cell biology, including BCR-triggered Ca2+ responses, impact on upregulation of B cell activation markers induced by multiple mechanisms of B cell activation and associated release of pro-inflammatory cytokines. The inventors found that clozapine and norclozapine suppressed BCR-promoted Ca2+ responses at higher concentrations, inhibited anti- lg/CD40-L/IL-21- and CpG/IL-15-stimulated expression of multiple B cell activation markers (CD71, CD80 and CD86) and exerted dose-dependent, functional inhibitory effects on B cell production of key cytokines implicated in the germinal centre response and/or inflammation: IL-6 and TNF-a.

Brief Description of the Drawings

Figure 1A-C. show the relative frequencies of numbers of patients at each serum concentration value for IgG, IgA and IgM respectively for clozapine-treated patients (black) and clozapine-naive patients (grey) (see Example 1).

Figure ID illustrates density plots showing the distribution of serum immunoglobulin levels in patients receiving clozapine referred for Immunology assessment (light grey left-most curve, n = 13) following removal of 4 patients (n=2 with haematological malignancy and n= 2 previously included within the inventor's recent case-control study (Ponsford et al 2018b)). Serum immunoglobulin distributions for clozapine-treated (mid-grey middle curve, n = 94) and clozapine-naive (dark grey right-most curve, n = 98) are also shown for comparison [adapted from (Ponsford et al., 2018b)]. Dotted lines represent the 5th and 95th percentiles for healthy adults (see Example 1).

Figure 2. shows the effect of duration of clozapine use on serum IgG levels (see Example 1).

Figure 3A. shows the number of class switched memory B cells (CSMB) (CD27+/lgM-/lgD-, expressed as a percentage of total CD19+ cells) in healthy controls, in patients taking clozapine referred to clinic and in patients with common variable immunodeficiency disorder (CVID) (see Example 1).

Figure 3B. shows B cell subsets, expressed as a percentage of total CD19 ⁺ cells, in patients with schizophrenia with a history of clozapine therapy referred to clinic (numbers as shown), common variable immunodeficiency (CVID, n=26) and healthy controls (n=17). B-cell subsets gated on CD19 ⁺ cells and defined as follows: Naive B-cells (CD27 lgD ⁺ lgM ⁺ ), Marginal Zone-like B-cells

(CD27 ⁺ lgD ⁺ lgM ⁺ ), Class-switched Memory B-cells (CD27 ⁺ lgD lgM ), and Plasmablasts

(CD19 ⁺ CD27 ^Hl lgD ). Non-parametric Mann-Whitney testing performed for non-normally distributed data, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 (see Example 1).

Figure 4A. shows the number of plasmablasts (CD38+++/lgM-, expressed as a percentage of total CD19+ cells) in healthy controls, in patients taking clozapine referred to clinic and in patients with common variable immunodeficiency disorder (CVID) (see Example 1).

Figure 4B. illustrates vaccine specific-lgG response assessment (see Example 1). Figure 5. shows gradual recovery of serum IgG post-discontinuation of clozapine from 3.5 to 5.95g/L over three years. LLN= lower limit of normal (see Example 1).

Figure 6A-C. shows interim data findings on the levels of circulating IgG, IgA and IgM in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right). Mean ± SEM (see Example 2).

Figure 7. shows interim data findings on peripheral blood levels of pneumococcal-specific IgG in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right). Mean ± SEM (see Example 2).

Figure 8A-B. shows interim data findings on peripheral blood levels of B cells (CD19 ⁺ ) in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right), expressed as absolute levels and as a percentage of lymphocytes (%, i.e. of T + B + NK cells). Mean ± SEM (see Example 2).

Figure 9A-C. shows interim data findings on peripheral blood levels of naive B cells (CD197CD27 ) in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right), expressed as a percentage of total B cells (CD19 ⁺ cells, %B), lymphocytes (%L), or absolute values (abs), respectively. Mean ± SEM (see Example 2).

Figure 10A-C. shows interim data findings on peripheral blood levels of memory B cells

(CD19 ⁺ /CD27 ⁺ ) in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right), expressed as a percentage of total B cells (CD19 ⁺ cells, %B), lymphocytes (%L), or absolute values (abs), respectively. Mean ± SEM (see Example 2).

Figure 11A-C. shows interim data findings on peripheral blood levels of class switched (CS) memory B cells (CD27 ⁺ /lgM /lgD ) in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right), expressed as a percentage of total B cells (CD19 ⁺ cells, %B), lymphocytes (%L), or absolute values (abs), respectively. Mean ± SEM (see Example 2).

Figure 12A-C. shows interim data findings on peripheral blood levels of IgM high IgD low

(CD27 ⁺ /lgM ⁺ VlgD ) memory B cells, i.e. post-germinal centre IgM only B cells, in patients on non clozapine antipsychotics ('control', left) versus clozapine (right), expressed as a percentage of total B cells (CD19 ⁺ cells, %B), lymphocytes (%L), or absolute values (abs), respectively. Mean ± SEM (see Example 2).

Figure 13A-C. shows interim data findings on peripheral blood levels of transitional B cells

(lgM ⁺⁺ /CD38 ⁺⁺ ) in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right), expressed as a percentage of total B cells (CD19 ⁺ cells, %B), lymphocytes (%L), or absolute values (abs), respectively. Mean ± SEM (see Example 2). Figure 14A-C. shows interim data findings on peripheral blood levels of marginal zone (MZ) B cells (CD27 ⁺ /lgD ⁺ /lgM ⁺ ) in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right), expressed as a percentage of total B cells (CD19 ⁺ cells, %B), lymphocytes (%L), or absolute values (abs), respectively. Mean ± SEM (see Example 2).

Figure 15A-C. shows interim data findings on peripheral blood levels of plasmablasts in patients on non-clozapine antipsychotics ('control', left) versus clozapine (right), expressed as a percentage of total B cells (CD19 ⁺ cells, %B), lymphocytes (%L), or absolute values (abs), respectively. Mean ± SEM (see Example 2).

Figure 16. shows the body weight growth curve of WT mice in response to clozapine at different doses versus haloperidol and vehicle controls. Mean ± SEM (see Example 3).

Figure 17. shows body weight comparisons of WT mice at days 3, 12 and 21 of treatment. Mean ± SEM (see Example 3).

Figure 18. shows the impact of clozapine versus haloperidol and vehicle control on overall B cell content and pre-pro B cell and pro B cell precursors in bone marrow of WT mice. Mean ± SEM (see Example 3).

Figure 19. shows the impact of clozapine versus haloperidol and vehicle control on pre-B cells, proliferating B cells and immature B cell precursors in bone marrow of WT mice. Mean ± SEM (see Example 3).

Figure 20. shows the impact of clozapine versus haloperidol and vehicle control on class-switched memory B cells, plasmablasts and long-lived plasma cells in bone marrow of WT mice. Mean ± SEM (see Example 3).

Figure 21. shows the impact of clozapine versus haloperidol and vehicle control on overall B cells, T cells, other cell populations (TCR- /B220 ) and activated T cells in spleen of WT mice. Mean ± SEM (see Example 3).

Figure 22. shows the impact of clozapine versus haloperidol and vehicle control on transitional (T1 and T2), follicular, marginal zone (MZ) and germinal centre (GC) B cells in spleen of WT mice. Mean ± SEM (see Example 3).

Figure 23. shows the impact of clozapine versus haloperidol and vehicle control on B cell subpopulations and T cells in the mesenteric lymph nodes (MLN) of WT mice. Mean ± SEM. T1 and T2, transitional type 1 and type 2 B cells, respectively. MZ, marginal zone. GC, germinal centre (see Example 3). Figure 24. shows the impact of clozapine versus haloperidol and vehicle control on circulating immunoglobulins in WT mice. Mean ± SEM (see Example 3).

Figure 25. shows impact of clozapine on day of clinical onset of CIA. Mean ± SEM (see Example 4).

Figure 26. shows impact of clozapine on incidence of CIA (see Example 4).

Figure 27. shows the impact of clozapine on the severity of CIA, judged by clinical score and thickness of first affected paw, in mice dosed from day 1 post-immunisation. Mean ± SEM (see Example 4).

Figure 28. shows the impact of clozapine on the severity of CIA, judged by number of affected paws by day of treatment with clozapine (day 15, D15 or day 1, Dl) post-immunisation. Mean ± SEM (see Example 4).

Figure 29. shows the impact of clozapine versus control on B220 ⁺ (i.e. CD45 ⁺ ) cells in spleen and local lymph node of CIA mice. Mean ± SEM (see Example 4).

Figure 30. shows the impact of clozapine versus control on plasma cells (PC) in spleen and local lymph node of CIA mice. Mean ± SEM (see Example 4).

Figure 31. shows the impact of clozapine versus control on germinal centre (GC) B cells (B220 ⁺ /lgD /Fas ⁺ /GL7 ⁺ ) in spleen and local lymph node of CIA mice. Mean ± SEM (see Example 4).

Figure 32. shows the impact of clozapine versus control on expression of GL7 on germinal centre (GC) B cells (B2207lgD /Fas7GL7 ⁺ ) in spleen and local lymph node of CIA mice. MFI, mean fluorescent intensity. Mean ± SEM (see Example 4).

Figure 33. shows the impact of clozapine versus control on peripheral blood anti-collagen IgGl and lgG2a antibody levels of CIA mice (see Example 4).

Figure 34. shows the impact of clozapine versus control on germinal centre resident T follicular helper cells (CD4 ⁺ PD1 ⁺ ) in spleen and local lymph node of CIA mice. Mean ± SEM (see Example 4).

Figure 35. shows the impact of clozapine versus control on expression of PD1 on germinal centre resident T follicular helper cells (CD4 ⁺ PD1 ⁺ ) in spleen and local lymph node of CIA mice. MFI, mean fluorescent intensity. Mean ± SEM (see Example 4).

Figure 36. shows the impact of clozapine versus control on expression of CXCR5 on germinal centre resident T follicular helper cells (CD4 ⁺ PD1 ⁺ ) in spleen and local lymph node of CIA mice. MFI, mean fluorescent intensity. Mean ± SEM (see Example 4). Figure 37. shows the impact of clozapine versus control on expression of CCR7 on germinal centre resident T follicular helper cells (CD4 ⁺ PD1 ⁺ ) in spleen and local lymph node of CIA mice. M FI, mean fluorescent intensity. Mean ± SEM (see Example 4).

Figure 38. shows protocol schematic for in vitro generation/differentiation of human plasma cells (see Example 5).

Figure 39. shows a schema of the healthy human volunteer study illustrating clozapine uptitration period followed by administration of typhoid vaccine (Typhim Vi) by injection (arrow) and then ongoing dosing with clozapine, as well as the Control cohort (vaccine only, no clozapine) (see Example 6).

Figure 40. shows the impact of clozapine versus control on Treg (CD4 ⁺ /CD25 ⁺ /FoxP3 ⁺ ) cells in spleen and local lymph node of CIA mice. Mean ± SEM (see Example 4).

Figure 41. shows the impact of clozapine versus control on expression of CD25 on Tregs in spleen and local lymph node of CIA mice. MFI, mean fluorescent intensity. Mean ± SEM (see Example 4).

Figure 42. shows the impact of clozapine versus control on expression of FoxP3 on Tregs in spleen and local lymph node of CIA mice. MFI, mean fluorescent intensity. Mean ± SEM (see Example 4).

Figure 43. shows geometric mean clozapine concentration time curve on a linear scale. Active = 41 days of clozapine dosing from Day -7 to Day 34 with a single intramuscular injection of Typhim Vi vaccine on Day 1; Lower Limit of Quantification = 30 pg/L. BLQ values were imputed as zero at pre dose and LLOQ/2 post-dose (see Example 6).

Figure 44. shows geometric mean desmethylclozapine (i.e. norclozapine) concentration-time curve on a linear scale. Active = 41 days of clozapine dosing from Day -7 to Day 34 with a single intramuscular injection of Typhim Vi vaccine on Day 1; Lower Limit of Quantification = 30 pg/L. BLQ values were imputed as zero at pre-dose and LLOQ/2 post-dose (see Example 6).

Figure 45. shows Individual Typhi Vi IgG antibody results. Active = 41 days of clozapine dosing from Day -7 to Day 34 with a single i.m injection of Typhim Vi vaccine on Day 1; Control = a single i.m injection of Typhim Vi vaccine on Day 1 (see Example 6).

Figure 46. shows arithmetic mean change from baseline IgG antibody levels (immunogenicity set). LLOQ = 0.17 g/L. Results reported as <LLOQ were imputed as LLOQ/2. Baseline defined as Day -7 pre- IM P for the active group and Day 1 pre-NIMP for the control group i.m = intramuscular, IMP = investigational medicinal product, LLOQ = lower limit of quantification, NIM P = non-investigational medicinal product (see Example 6). Figure 47A-B. shows Arithmetic Mean Change from Baseline IgM Antibody Levels (Immunogenicity Set) and Arithmetic Mean (±SE) Change from Baseline IgM antibody levels (Per Protocol Set) (see Example 6).

Figure 48. shows correlation analysis of change from baseline CD19+ B cells vs plasma clozapine (immunogenicity set) (see Example 6).

Figure 49. shows correlation analysis of change from baseline lgD+ CD27+ B cells vs plasma clozapine (immunogenicity set) (see Example 6).

Figure 50A-B. shows Individual Flow Cytometry Analysis B Cell Panel Results (Safety Set) for IgM hi IgD lo memory B cells (xlO ^A 9/L), and IgM hi IgD lo memory B cells (% B), both excluding subject 015, Day 7 result (see Example 6).

Figure 51A-D. shows absolute and normalised spleen weights at Day 14 and Day 28. CLZ = clozapine; NDMC = N-desmethylclozapine (i.e. norclozapine); CsA = ciclosporin A; *= P<0.05, **=P<0.01 and ****P _< 0.0001 for the indicated comparison or versus vehicle where no comparison line is depicted (see Example 7).

Figure 52. shows serum anti-TNP IgM levels at Day 7 post-immunisation with KLH-TNP. *= P<0.05, and * * * * p<0.0001 versus vehicle (see Example 7).

Figure 53A. shows the profile of serum anti-TNP IgG over time in response to primary immunisation with KLH-TNP (at Day 0) and repeat (booster) immunisation at Day 14 (see Example 7).

Figure 53B-E show serum anti-TNP IgM levels at Day 7, 14, 21 and 28 post-immunisation with KLH- TNP. *= P<0.05, **=P<0.01, ***=P<0.001 and ****P<0.0001 for the indicated comparison or versus vehicle where no comparison line is depicted (see Example 7).

Figure 54A-D. shows donor average calcium signalling. Data presented as (A) peak response, (B) area under the curve (AUC), (C) % responding cells or (D) slope of the response. Data presented as mean+SEM arising from six independent donors, except for clozapine and norclozapine (NDMC) each at 10 pg/mL) which represent a single data point and clozapine and norclozapine (each at 0.01 pg/mL) which represents mean+SEM arising from five independent donors. CLZ = clozapine; NDMC = N-desmethylclozapine (i.e. norclozapine) (see Example 8).

Figure 55A-C. shows donor average % of expression of the activation markers CD71 (A), CD80 (B) or CD86 (C) of total B cells incubated for 1 hour at 37°C in the presence of clozapine, norclozapine, ibrutinib, BAY-61-3606 or chloroquine in the absence (vehicle 0.1% DMSO or 0.1% DH20) or media alone, at the indicated concentrations. B cells were then cultured in the presence of: media (unstimulated), anti-lg + CD40L + IL-21, polymeric anti-lgM or CpG-ODN + IL-15 for 5 days (see Example 8).

Figure 56. shows donor average IL-6 levels from B cells incubated for 1 hour at 37°C in the presence of clozapine, norclozapine, ibrutinib, BAY-61-3606 or chloroquine in the absence (vehicle 0.1% DMSO or 0.1% DH20) or media alone, at the indicated concentrations. B cells were then cultured in the presence of: media (unstimulated, data not shown), anti-lg + CD40L + IL-21, polymeric anti-lgM or CpG-ODN + IL-15 for 5 days. Levels of IL-6 were quantified in the supernatant by Luminex assay. Data presented as mean+SEM arising from six independent donors, except for clozapine and norclozapine (each at 0.01 pg/mL) which represents mean+SEM arising from five independent donors. - indicates at least 1 donor below limit of detection for IL-6 (0.32 pg/mL) (see Example 8).

Figure 57. shows donor average TNF-a levels from B cells incubated for 1 hour at 37°C in the presence of clozapine, norclozapine, ibrutinib, BAY-61-3606 or chloroquine in the absence (vehicle 0.1% DMSO or 0.1% DH20) or media alone, at the indicated concentrations. B cells were then cultured in the presence of: media (unstimulated, data not shown), anti-lg + CD40L + IL-21, polymeric anti-lgM or CpG-ODN + IL-15 for 5 days. Levels of TNF-a were quantified in the supernatant by Luminex assay. Data presented as mean+SEM arising from six independent donors, except for clozapine and norclozapine (each at 0.01 pg/mL) which represents mean+SEM arising from five independent donors. - indicates at least 1 donor below limit of detection for TNF-a (0.56 pg/mL) (see Example 8).

Detailed description of the invention

The present invention also provides a method of treatment or prevention of a pathogenic immunoglobulin driven B cell disease, a pathogenic immunoglobulin driven B cell disease with a T cell component or a pathogenic IgE driven B cell disease in a subject by administering to said subject an effective amount of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof, in particular, wherein said compound causes mature B cells to be inhibited in said subject. The compound may also act on the B cell receptor (BCR) by suppressing BCR-promoted Ca2+ responses. Thus, the compound may suppress a BCR-promoted Ca2+ response.

The present invention also provides use of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof in the manufacture of a medicament for the treatment or prevention of a pathogenic immunoglobulin driven B cell disease, a pathogenic immunoglobulin driven B cell disease with a T cell component or a pathogenic IgE driven B cell disease in a subject, in particular, wherein said compound causes mature B cells to be inhibited in said subject. The compound may also act on the B cell receptor (BCR) by suppressing BCR-promoted Ca2+ responses. Thus, the compound may suppress a BCR-promoted Ca2+ response.

Clozapine or norclozapine or pegylated derivatives thereof may optionally be utilised in the form of a pharmaceutically acceptable salt and/or solvate and/or prodrug. In one embodiment of the invention clozapine or norclozapine or pegylated derivatives thereof is utilised in the form of a pharmaceutically acceptable salt. In a further embodiment of the invention clozapine or norclozapine is utilised in the form of a pharmaceutically acceptable solvate. In a further embodiment of the invention clozapine or norclozapine or pegylated derivatives thereof is not in the form of a salt or solvate. In a further embodiment of the invention clozapine or norclozapine or pegylated derivatives thereof is utilised in the form of a prodrug. In a further embodiment of the invention clozapine or norclozapine or pegylated derivatives thereof is not utilised in the form of a prodrug. In another embodiment of the invention clozapine is utilised in the form of a

pharmaceutically acceptable salt. In a further embodiment of the invention norclozapine is utilised in the form of a pharmaceutically acceptable salt. In another embodiment of the invention pegylated clozapine is utilised in the form of a pharmaceutically acceptable salt. In another embodiment of the invention pegylated norclozapine is utilised in the form of a pharmaceutically acceptable salt. In a further embodiment of the invention clozapine is utilised in the form of a pharmaceutically acceptable solvate. In a further embodiment of the invention norclozapine is utilised in the form of a pharmaceutically acceptable solvate. In a further embodiment of the invention pegylated clozapine is utilised in the form of a pharmaceutically acceptable solvate. In a further embodiment of the invention pegylated norclozapine is utilised in the form of a

pharmaceutically acceptable solvate. In a further embodiment of the invention clozapine is not in the form of a salt or solvate. In a further embodiment of the invention norclozapine is not in the form of a salt or solvate. In a further embodiment of the invention pegylated clozapine is not in the form of a salt or solvate. In a further embodiment of the invention pegylated norclozapine is not in the form of a salt or solvate. In a further embodiment of the invention clozapine is utilised in the form of a prodrug. In a further embodiment of the invention is utilised in the form of a prodrug. In a further embodiment of the invention pegylated clozapine is utilised in the form of a prodrug. In a further embodiment of the invention pegylated norclozapine is utilised in the form of a prodrug. In a further embodiment of the invention clozapine is not utilised in the form of a prodrug. In a further embodiment of the invention is not utilised in the form of a prodrug. In a further embodiment of the invention pegylated clozapine is not utilised in the form of a prodrug. In a further embodiment of the invention pegylated norclozapine is not utilised in the form of a prodrug.

The term "pathogenic immunoglobulin driven B cell disease" includes B cell mediated disease, especially autoimmune disease, which involves pathogenic immunoglobulins (e.g. IgG, IgA and/or IgM) targeting a self-antigen (e.g. auto-antibody IgG, IgA and/or IgM) as a principal mechanism.

Suitably the pathogenic immunoglobulin driven B cell disease is a pathogenic IgG driven B cell disease. Alternatively, suitably it is a pathogenic IgA driven B cell disease.

The term "pathogenic IgG driven B cell disease" includes B cell mediated disease, especially autoimmune disease, which involves pathogenic IgG targeting a self-antigen (i.e. auto-antibody IgG) as a principal mechanism.

The term "pathogenic IgA driven B cell disease" includes B cell mediated disease, especially autoimmune disease, which involves pathogenic IgA targeting a self-antigen (i.e. auto-antibody IgA) as a principal mechanism.

The term "pathogenic immunoglobulin B cell disease with a T cell component" includes B cell mediated disease, especially autoimmune disease, which involves pathogenic immunoglobulin (e.g. IgG, IgA and/or IgM) targeting a self-antigen (e.g. auto-antibody IgG, IgA and/or IgM) and with T cell mediated inflammation as a principal mechanism and/or abnormal or excessive T _F H function as a mechanism, promoting primary T-dependent B cell autoreactive responses. The term also includes immune rejection of an allograft as in graft versus host disease.

The term "pathogenic IgE driven B cell disease" includes B cell mediated disease, especially inflammatory disease, which involves exogenous antigens causing abnormally high and pathogenic IgE levels as a principal mechanism.

The range of self-antigens involved in autoimmune diseases include desmoglein 3, BP180, BP230, (pemphigus), dystonin and/or type XVII collagen (pemphigoid), myelin (multiple sclerosis), pancreatic beta cell proteins (Type 1 diabetes mellitus), nicotinic acetylcholine receptors

(myaesthenia gravis), neuronal surface proteins (autoimmune epilepsy and encephalitis), fibrillarin (scleroderma), anti-dsDNA and cardiolipin (systemic lupus erythematosus), 2-hydrolase

(autoimmune Addison's disease), FceRI (chronic autoimmune urticaria) and acetylcholine receptor (myasthenia gravis). The range of self-antigens involved in pathogenic IgA driven B cell diseases include tissue transglutaminase (dermatitis herpetiformis and coeliac disease), gliadin IgA (coeliac disease) and dystonin and/or type XVII collagen (linear IgA disease).

Exemplary pathogenic IgG driven B cell diseases are autoimmune diseases including those which may be selected from the group consisting of the skin related diseases pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, autoimmune alopecia, vitiligo, dermatitis herpetiformis and chronic autoimmune urticaria. Alternatively, the disease may be the gut related disease coeliac disease. Alternatively, the diseases may be selected from the group consisting of the thyroid gland related diseases Graves' disease and Hashimoto's thyroiditis.

Alternatively, the diseases may be the pancreas related disease Type 1 diabetes mellitus.

Alternatively, the disease may be the adrenal gland related disease autoimmune Addison's disease. Alternatively, the diseases may be selected from the group consisting of the haematological related diseases autoimmune haemolytic anaemia, autoimmune thrombocytopenic purpura and cryoglobulinemia. Alternatively, the disease may be the gut related disease pernicious anaemia. Alternatively, the diseases may be selected from the group consisting of the neurological related diseases myasthenia gravis, multiple sclerosis, neuromyelitis optica and autoimmune epilepsy and encephalitis. Alternatively, the diseases may be selected from the group consisting of the liver related diseases autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis. Alternatively, the diseases may be selected from pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, chronic autoimmune urticaria, Graves' disease, Hashimoto's thyroiditis, Type 1 diabetes mellitus, autoimmune haemolytic anaemia, autoimmune thrombocytopenic purpura, cryoglobulinemia, myasthenia gravis, neuromyelitis optica, autoimmune epilepsy and encephalitis, autoimmune hepatitis, primary biliary cirrhosis, linear IgA disease and IgA nephropathy. Alternatively, the diseases may be selected from pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, chronic autoimmune urticaria, Graves' disease, Hashimoto's thyroiditis, Type 1 diabetes mellitus, autoimmune haemolytic anaemia, autoimmune thrombocytopenic purpura, cryoglobulinemia, myasthenia gravis, neuromyelitis optica, autoimmune epilepsy and encephalitis, autoimmune hepatitis and primary biliary cirrhosis. Alternatively, the diseases may be selected from of pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid. Alternatively, the diseases may be selected from dermatitis herpetiformis, linear IgA disease, IgA nephropathy, pemphigus vulgaris, pemphigus foliaceus, cicatricial pemphigoid and bullous pemphigoid.

Exemplary pathogenic immunoglobulin driven B cell diseases with a T cell component may be the skin related diseases vitiligo, psoriasis, coeliac disease, dermatitis herpetiformis or discoid lupus erythematosus. Alternatively, the disease may be the muscle related diseases dermatomyositis or polymyositis. Alternatively, the disease may be the pancreas related disease Type 1 diabetes mellitus. Alternatively, the disease may be the adrenal gland related disease autoimmune Addison's disease. Alternatively, the disease may be the neurological related disease multiple sclerosis.

Alternatively, the disease may be the lung related disease interstitial lung disease. Alternatively, the disease may be the bowel related diseases Crohn's disease or ulcerative colitis. Alternatively, the disease may be the thyroid related disease thyroid autoimmune disease. Alternatively, the disease may be the eye related disease autoimmune uveitis. Alternatively, the disease may be the liver related diseases primary biliary cirrhosis or primary sclerosing cholangitis. Alternatively, the disease may be undifferentiated connective tissue disease. Alternatively, the disease may be an immune- mediated inflammatory disease (IMID) such as scleroderma, rheumatoid arthritis or Sjogren's disease. Alternatively, the disease may be autoimmune thrombocytopenic purpura. Alternatively, the disease may be a connective tissue disease such as systemic lupus erythematosus. Alternatively, the disease may be mixed connective tissue disease (MCTD). Alternatively, the disease may be graft versus host disease.

The range of exogenous antigens for pathogenic IgE driven B cell diseases include neutrophils (Churg-Strauss vasculitis) and pollen antigens (allergic rhinitis, allergic eye disease and atopic asthma, although there may be other causes).

Exemplary pathogenic IgE driven B cell diseases may be the lung related disease atopic asthma. Alternatively, the disease may be the skin related diseases atopic dermatitis and chronic non- autoimmune urticaria. Alternatively, the disease may be the neurological related disease Churg- Strauss vasculitis. Alternatively, the disease may be the nasal related disease allergic rhinitis.

Alternatively, the disease may be the eye related disease allergic eye disease. Alternatively, the disease may be the oesophagus related disease eosinophilic oesophagitis.

References highlighting the role of B cells, T cells and pathogenic IgE antibodies in the

aforementioned diseases are provided below:

Pemphigus vulgaris and pemphigus foliaceus

Pemphigus is a B cell-mediated autoimmune blistering disease of the skin and mucosa characterised by the generation of pathogenic autoantibodies, predominantly of the lgG4 subclass (but also IgGl and less so IgA) (Futei et al., 2001), against desmogleins (DSG3 and DSG1, and occasionally desmocollin 3) resulting in acanthloysis (Kasperkiewicz et al., 2017). Classical pemphigus involves IgG autoantibodies, but mixed IgG/lgA and IgA forms are recognised (Hegazy et al., 2016; Toosi et al., 2016). Pemphigus vulgaris is characterised by anti-DSG3 antibodies with/without anti-DSGl, while patients with pemphigus foliaceus exhibit anti-DSGl antibodies.

Circulating pathogenic IgG autoantibodies from patients with pemphigus have been shown to disrupt keratinocyte monolayers in vitro (Di Zenzo et al., 2012) and to result in pemphigus-like disease lesions on passive transfer to neonatal mice in vivo (Anhalt et al., 1982), an effect that was dose-dependent. Furthermore, disease activity correlates with anti-DSG3 autoantibody titre (Ishii et al., 1997). Passive transplacental transfer of pathogenic autoantibodies from mothers with pemphigus vulgaris has been reported to result in characteristic skin lesions which resolve spontaneously (Ruach et al., 1995). Accordingly, in pemphigus pathogenic immunoglobulin is both necessary and sufficient to induce disease. Substantiating the key role of B cells in the pathogenesis of pemphigus, B cell depletion using rituximab is clinically effective (Joly et al., 2007), with patients in complete remission characterised by near complete disappearance of desmoglein-specific circulating lgG+ B cells and serum anti-DSG antibodies (Colliou et al., 2013). Furthermore, Dsg-1 and Dsg-3 specific (i.e. antigen specific) B cells have been identified in pemphigus skin lesions, together with lgG ⁺ plasma cells (Takahashi, 2017).

Bullous pemphigoid and cicatricial pemphigoid

Bullous pemphigoid is characterised immunologically by the presence of circulating IgG

autoantibodies targeting the dermatoepidermal basement membrane zone, particularly the NC16a domain of the BP180 autoantigen (Diaz et al., 1990). Serum titres of anti-NC16a antibodies correlate with disease severity (Schmidt et al., 2000) and cause blister formation either directly or through complement fixation. Notably, circulating antigen-specific autoreactive plasmablasts and memory B cells specific for bullous pemphigoid autoantigens have been identified in patients with pemphigoid (Laszlo et al., 2010), with the latter able to differentiate into antibody secreting cells and produce anti-NC16a specific IgG antibodies in vitro (Leyendeckers et al., 2003). BAFF (B-cell activating factor belonging to the tumour necrosis factor (TNF) family), a key regulator of B cell survival and proliferation (including of autoreactive B cells) and thought to play a key role in the induction of autoimmune disease primarily or substantially mediated by B cells, is significantly elevated in serum of patients with bullous pemphigoid and decreases with treatment (Asashima et al., 2006).

Moreover, BAFF has been identified on naive and memory B cells in bullous pemphigoid but not healthy controls, where it may function as an autocrine factor promoting survival of autoreactive B cells (Qian et al., 2014). Further evidence for an environment conducive to B cell activation is the observation of elevated levels of circulating soluble CD40 ligand (sCD40L) in patients in bullous pemphigoid, particularly early at disease onset and in association with recurrences (Watanabe et al., 2007). Analogous findings have been observed in other autoimmune diseases such as SLE. The interaction between CD40 (on B cells and other antigen-presenting cells) and CD40L (appearing transiently on CD4 ⁺ T cells) is required for B cell differentiation and immunoglobulin class switching.

Substantiating a key pathogenic role for B cells in bullous pemphigoid, B cell depletion with rituximab has clinical efficacy in pemphigoid, including in recalcitrant and cicatricial cases (Li et al., 2011) with greater effect noted in IgG-dominant cases (Lamberts et al., 2018). Highlighting the importance of antibody secreting cell populations unaffected by rituximab therapy, persistence of autoreactive IgA-secreting plasmablasts/plasma cells has been described in association with refractory pemphigoid (He et al., 2015). Immunoapheresis, a therapeutic approach which removes immunoglobulins and immune complexes, has shown efficacy in severe/life threatening

autoimmune bullous disease, including pemphigoid gestationis and pemphigus, in association with reduction in pathogenic immunoglobulin (Marker et al., 2011).

Autoimmune alopecia (autoimmune hair loss; autoimmune alopecia areata)

Alopecia areata (AA) is a common disorder characterised by acute onset of non-scarring hair loss, most frequently in patches affecting the scalp, but can involve hair loss of the entire scalp (alopecia totalis), facial hair (including eyebrows, eyelashes, beard), or loss of entire scalp and body hair (alopecia universalis) (Islam et al., 2015). Alopecia areata is thought to reflect an organ-specific autoimmune disease of the hair follicle (Trueb and Dias, 2018).

Supporting an autoimmune basis for AA, patients often develop or have a history of other canonical autoimmune disease, including SLE, vitiligo, autoimmune thyroid disease, myaesthenia gravis and rheumatoid arthritis (Islam et al., 2015). While there is a recognised prominent CD8 ⁺ T cell-driven component directed against anagen-stage hair follicles (Guo et al., 2015), the pathobiology of AA is not fully understood. Plasma cells have been described in the peribulbar inflammatory infiltrate accompanying AA in patients (Elston et al., 1997; Ranki et al., 1984) and, using transmission electron microscopy, active plasma cells have been identified in acute AA (McElwee et al., 2013). Similar dermal observations together with hair follicle specific IgG on direct immunofluorescence have been noted in dogs exhibiting an AA homologue (Tobin et al., 2003). Antibodies to antigens selectively expressed in hair follicles of patients with AA have been identified (Tobin et al., 1994b). Circulating autoantibodies against hair follicle-specific keratins have also been described in C3H/HeJ mice with AA-like hair loss (Tobin et al., 1997). In contrast to the low levels of primarily IgM anti-hair follicle antibodies identified in normal individuals(Tobin et al., 1994a), those associated with AA are much higher in titre, not present in healthy individuals and of the IgG subclass, suggesting a class-switching as an important process in the immunopathogenesis of AA. Notably autoantibodies precede disease onset in the C3H/HeJ mouse model of AA, suggesting that the autoantibodies detected are not merely a secondary response to damage of hair follicles (Tobin, 2003). Notably, hair follicle autoantibody profile is modulated by topical therapy with diphencyprone used for AA, with very significant reductions in the titre of IgG anti-hair follicle antibodies in patients with complete and sustained hair regrowth, indicating that such autoantibody levels correlate with disease activity (Tobin et al., 2002). Supporting the pathogenic potential of such autoantibodies in AA, passive transfer of equine IgG fractions from a horse affected with AA-like hair loss to the anagen skin of wildtype C57BL mice disrupted hair regrowth around the site of injection, including up to 13 weeks post-injection, a finding not observed after injection of normal equine IgG (Tobin et al., 1998).

Autoimmune thyroid disease (AITD), including Graves' disease and Hashimoto's thyroiditis

AITD is an organ-specific autoimmune disorder characterised by breakdown of self-tolerance to thyroid antigens. Genome-wide association studies have revealed a role for genetic variants in B cell signalling molecules in the development of AITD (Burton et al., 2007), including FCRL3 (Chu et al., 2011b) and BACH2 involved in B cell tolerance, maturation and class switching (Muto et al., 2004).

Pathologically, AITD exhibits intense lymphocyte accumulation in the thyroid gland, including B cells at the time of diagnosis (notably in Hashimoto's thyroiditis) and production of anti-thyroid antibodies (Zha et al., 2014). Patients with recent-onset AITD display thyroid antigen-reactive B cells in the peripheral blood which are no longer anergic but express the activation marker, CD86, consistent with activation of these cells to drive autoantibody production (Smith et al., 2018).

Graves' disease is characterised by production of pathognomonic agonistic anti-thyrotropin receptor IgG autoantibodies (found in 80-100% of untreated patients) which mimic TSH and stimulate thyroid hormone overproduction and thyroid enlargement (Singh and Hershman, 2016). Patients with Graves' disease exhibit elevated transitional and pre-naive mature B cells in peripheral blood, with levels positively correlating with those of free thyroxine (Van der Weerd et al., 2013). Consistent with a B cell-driven pathophysiological process and potentially contributing to the expansion of these B cell populations, the serum levels of BAFF (B lymphocyte activating factor) - a key factor promoting B cell autoantibody production by increasing B cell survival and proliferation - are raised in patients with Graves' disease and fall in response to methylprednisolone treatment (Vannucchi et al., 2012). Hyperthyroidism itself promotes plasma cytogenesis to increase plasma cells in the bone marrow (Bloise et al., 2014). B cell depletion using anti-mouse monoclonal CD20 antibody in a mouse immunisation model of model of Graves' disease is effective in suppressing anti-TSHR antibody generation and hyperthyroidism given before immunisation or 2 weeks later (Ueki et al., 2011). Mirroring this, rituximab has demonstrated efficacy clinically in Graves' orbitopathy (Salvi et al., 2013).

In Hashimoto's thyroiditis, B cells generate autoantibodies against thyroglobulin (>90% patients) and thyroid peroxidase which lead to apoptosis of thyroid follicular cells via antibody-dependent cell- mediated cytotoxicity. Plasma cell accumulation has been noted in thyroidectomy specimens from patients with Hashimoto's thyroiditis in association with foci of thyroid follicular destruction (Ben- Skowronek et al., 2013).

TFH cells, which regulate (auto-)antibody production by B cells, are found to be expanded in the circulation of patients with AITD, with a positive correlation with autoantibody titres and also levels of free thyroid hormone in Graves' disease; moreover, these cells reduce with therapy and have been found to be enriched in thyroid tissue from patients with Hashimoto's thyroiditis (Zhu et al.,

2012).

Autoimmune haemolytic anaemia (AIHA)

AIHAs are autoimmune disorders characterised by pathogenic autoreactive antibodies against red blood cells leading to reduced red cell survival and anaemia (Garvey, 2008). Anti-red blood cell antibodies in these conditions are central to the destruction of red blood cells via either direct lysis (through complement activation) or antibody-dependent cytotoxicity (Barcellini, 2015).

In warm AIHA (wAIHA) this is mediated via macrophage FcyR recognition of IgG-coated red blood cells (polyclonal and of the IgGl isotype, less so lgG3) and progressive membrane removal with ultimate formation of spherocytes which are trapped in splenic sinusoids and removed (LoBuglio et al., 1967). These autoantibodies are generated through the activation of autoreactive B cell clones (Fagiolo, 2004). Further evidence of a central role for B cells in AIHA comes from trial findings that B cell depletion with rituximab, in combination with glucocorticoid, shows efficacy over glucocorticoid alone in terms of both rate and duration of response (Birgens et al., 2013). Notably, active B cell responses are present in spleens of wAIHA patients, including large numbers of germinal centre B cells and plasmablasts/plasma cells which secrete anti-red blood cell antibodies (Mahevas et al., 2015). Patients with newly diagnosed wAIHA display an expansion of circulating GC-derived plasmablasts, more commonly IgG-secreting. Both splenic GC B cells and circulating plasmablasts reduce with corticosteroid therapy, indicating that the spleen is involved in both the destruction of red blood cells and the generation of autoreactive antibodies; as a corollary, splenectomy is associated with a durable response in many patients (Mahevas et al., 2015). Cryoglobulinaemia, refers to the presence of cryoglobulins in the serum; these are immunoglobulins which precipitate in vitro below 37°C and heterogeneous in composition (IgM, IgG or both). They result from mono- or poly-clonal B cell expansion, typically in association with lymphoproliferative disease, chronic infection or autoimmune disease (Ramos-Casals et al., 2012). In the setting of hepatitis C virus (HCV) infection, cryoglobulinaemia can occur as a B cell proliferation disorder and lead to systemic vasculitis through generation of monoclonal IgM which cross-reacts with immunoglobulins directed against HCV core proteins (Knight et al., 2010). Cryoglobulins are pathogenic through their ability to precipitate in the microcirculation and to induce immune- complex-mediated inflammatory injury. While treatment is focused on the underlying cause (e.g. antiviral therapy) combined with generalised immunosuppression, both plasma exchange and plasmapheresis (Payet et al., 2013) are effective in removing cryoglobulins in severe cases (Rockx and Clark, 2010). Highlighting the importance of B cells in the disease process, B cell depletion with rituximab is an effective therapeutic strategy in cryoglobulinaemic vasculitis (De Vita et al., 2012).

Pernicious anaemia (PA)

This refers to a megaloblastic anaemia due to impaired vitamin B12 absorption resulting from immune destruction of gastric parietal cells (which produce intrinsic factor required for B12 absorption) in the setting of atrophic gastritis (Bedeir et al., 2010). PA is characterised by circulating anti-parietal cell antibodies (~90% patients) of IgG, IgA and IgM isotypes and anti-intrinsic factor antibodies (~60%), the latter circulating IgG in class and specific markers for PA (Bizzaro and Antico, 2014). While autoimmune gastritis is thought of as a primarily T cell mediated disease, these autoantibodies are thought to contribute to the pathogenesis of PA. Specifically, parietal cell antibodies have been argued to promote destruction of gastric parietal cells based on preclinical studies administering these to rats (Tanaka and Glass, 1970), with demonstration of IgG antibodies on the surface of and within parietal cells suggesting access to the H ⁺ /K ⁺ ATPase (Burman et al., 1992). Notably parietal cell autoantibodies can predict development of overt atrophic gastritis (Tozzoli et al., 2010). In the case of intrinsic factor, while circulating autoantibodies are IgG in class, those secreted into gastric juice are IgA and thought to contribute to progression of autoimmune gastritis to PA (Osborne and Sobczynska-Malefora, 2015). Intrinsic factor antibodies can block the binding of cobalamin to intrinsic factor, or to block the binding of the intrinsic factor-cobalamin complex to its receptor in the ileum (Rowley and Whittingham, 2015). Recently, an increase in lgG4+ plasma cells has recently been identified in gastric mucosa of PA patients and not observed in other types of gastritis, suggesting specific involvement of these cells in the disease process (Bedeir et al., 2010). Myaesthenia gravis (MG)

In MG, IgG autoantibodies directed against the nicotinic acetylcholine receptor (in ~85% of patients) or other synaptic antigens (muscle-specific kinase and low-density lipoprotein receptor-related protein 4) present at the neuromuscular junction result in skeletal muscle weakness. The autoantibodies affect the function of these antigens to induce disease via multiple mechanisms, including complement-mediated membrane destruction (Engel and Arahata, 1987), antigenic modulation (e.g. cross-linking by bivalent IgGl and lgG3 to result in internalisation of AChR to reduce the available cell surface pool) (Drachman et al., 1978), ligand binding site competition (e.g. with ACh) (Drachman et al., 1982) and potentially steric hindrance (Huijbers et al., 2014).

Mirroring early observations of the ability of patient-derived immunoglobulin fraction of serum to induce disease in mice (Toyka et al., 1975), direct isolation and analysis of the anti-AChR antibody repertoire from peripheral memory B cells of patients with MG has identified pathogenic antibody (B12L) which induces a myasthenic phenotype in rats upon single dose passive transfer, with evidence of dose-dependency (Makino et al., 2017).

In addition to producing pathogenic immunoglobulin, multiple lines of evidence confirm the fundamental importance of B cells in the immunopathogenesis of MG. A deformed naive and memory B cell repertoire has been identified consistent with defective tolerance checkpoints in the naive compartment (Vander Heiden et al., 2017). Other observations indicate increased frequency of newly emigrant or transitional B cells and mature naive B cells with autoreactive B cell receptors, further indicating defective central (i.e. bone marrow) tolerance mechanisms in MG (Lee et al., 2016a). These, coupled with the presence of additional autoantibody specificities and high frequency of a second autoimmune disease in such patients, highlight the importance of dysregulated B cell self-tolerance in the pathogenesis of MG.

Studies of thymic populations from patients with MG have revealed B cells organised in germinal centres which are activated (Leprince et al., 1990), with thymic lymphocytes able to synthesis anti- AChR antibody (Vincent et al., 1978). As a corollary, thymectomy is associated with clinical improvement associated with a fall in autoantibody titre (Vincent et al., 1983; Wolfe et al., 2016). In addition to thymus, pathogenic antibody secreting cells have been identified in lymph nodes (Fujii et al., 1985a) and the bone marrow of patients with MG (Fujii et al., 1985b). Patients with MG have been observed to feature an expanded circulating plasmablast/plasma cell pool (Kohler et al., 2013).

Neuromyelitis optica (NMO) NMO is a demyelinating disorder of the central nervous system (CNS) typically presenting with recurrent episodes of optic neuritis and transverse myelitis. The majority (~75%) of patients exhibit IgG autoantibodies against glial aquaporin-4 (AQP4) water channels (Bennett et al 2015).

Intracerebral co-injection of IgG from AQP4 positive NMO patients with human complement into mice recapitulates key aspects of NMO histology, including loss of AQP4 expression, glial cell oedema, breakdown of myelin, cerebral oedema and neuronal cell death (Saadoun et al., 2010). Critically supporting a direct pathogenic role for these autoantibodies in mediating CNS injury, these features were not observed when IgG from non-NMO patients was used, or injection of IgG from NMO patients into AQP4-null mice (Saadoun et al., 2010). Plasmablasts are expanded in the peripheral blood of patients with NMO, capable of producing anti-AQP4 autoantibodies, with IgG plasmablasts enriched in cerebrospinal fluid (CSF) lymphocytes during NMO relapses (Chihara et al., 2013). Furthermore, IgG plasmablasts from peripheral blood and CSF of patients with NMO exhibit high frequencies of mutations in complementarity-determining regions (CDR) consistent with a post- germinal centre lineage and share CDR sequences suggesting migration of plasmablasts from periphery to the CSF to promote local autoantibody production (Chihara et al., 2013). Indeed, peripheral blood plasmablasts have been shown to be the primary producers of anti-AQP4 antibodies in the blood, further increased during relapses and promoted by IL-6 whose levels are increased in NMO (Chihara et al., 2011). Depletion of B cells using rituximab reduces NMO relapse frequency in patients (Damato et al., 2016).

Autoimmune epilepsy syndromes and autoimmune encephalitis

The autoimmune epilepsy syndromes are immune-mediated disorders characterised by recurrent, uncontrolled seizures which are often anti-epileptic drug resistant (Britton, 2016). While seizures are a recognised feature of autoimmune encephalitis and multifocal paraneoplastic disorders, they are increasingly recognised in the absence of typical syndromic features of encephalitis, i.e. as a distinct entity (Britton, 2016).

Autoantibodies against neural antigens such as voltage-gated potassium channel (VGKC) complex proteins, glycine receptors, glutamate/AMPA receptor subtype 3, glutamic acid decarboxylase (GAD), N-methyl-D-aspartate (NMDA) receptors (NMDAR), collapsin response-mediator protein 5 and ganglionic acetylcholine receptor are well-described in cohorts of patients with epilepsy including in those newly diagnosed and frequently resistant to conventional anti-epileptic drugs (Brenner et al., 2013; Ganor et al., 2005; McKnight et al., 2005; Quek et al., 2012). Supporting an immune basis for the manifestations, such cases have been reported to respond well to

immunotherapy including IV immunoglobulin and plasmapheresis (Quek et al., 2012). Clear correlation between autoantibodies and clinical seizures have been identified such as for GABA in limbic encephalitis with seizures (Lancaster et al., 2010) and NMDA in the context of anti-NMDA receptor encephalitis (Dalmau et al., 2008).

A large body of evidence supports a key role for B cells and the pathogenicity of autoantibodies in autoimmune encephalitis. The neuronal pathology of autoimmune encephalitis includes evidence of immunoglobulin on the surface of neurons (e.g. anti-VGKC-complex encephalitis), together with infiltration of CD20+ B cells and CD138+ plasma cells, supporting a B cell-mediated disease mechanism, particularly in those encephalitides with antibodies directed against surface antigens (Bien et al., 2012). Patients with treatment-naive autoimmune NMDAR encephalitis exhibit intrathecal (i.e. within CSF) B cell and plasma cell accumulation and intrathecal anti-NMDAR IgG antibody production (Malviya et al., 2017). Moreover, both intrathecal B cell and plasma cell accumulation correlate well with disease course and reflect response to immunotherapy (Malviya et al., 2017).

Hippocampal neurons cultured with CSF or purified IgG containing autoantibodies against NMDA from patients with NMDAR encephalitis results in reduced surface NMDAR cluster expression in a titre-dependent manner via cross-linking and internalisation of the receptors (Hughes et al., 2010). Consistent with an impact on function, patients' antibodies selectively reduce NMDAR currents of cultured rat hippocampal neurons (Hughes et al., 2010).

Passive transfer of cerebrospinal fluid (CSF) from patients with NMDAR encephalitis into the cerebral ventricles of wildtype C57BL6/J mice results in progressive memory deficits, anhedonic and depressive like behavioural changes which worsen over 14 days and resolve upon discontinuation of infusion, effects not seen with control CSF (Planaguma et al., 2015). Histologically these clinical features were accompanied by progressive elevation in brain-bound anti-NMDAR antibodies, largely in the hippocampus, and reduced surface density of NMDAR (Planaguma et al., 2015). Conversely, reversibility and recovery were associated with a fall in brain-bound antibody levels and recovery of NMDAR concentration. Further implicating NMDAR encephalitis as a humorally driven autoimmune disease, single recombinant human NMDAR-specific monoclonal antibody reconstructed from patient-derived clonally expanded intrathecal plasma cells is sufficient to recapitulate key features of NMDAR encephalitis in vitro and in vivo (Malviya et al., 2017).

Clinically, autoantibody levels in patients with autoimmune encephalitis (such as anti-NMDA receptor encephalitis) correlate with neurological outcome, with antibody levels in CSF more closely correlated with relapses than levels in serum (Gresa-Arribas et al., 2014). Further supporting a pathogenic role for autoantibodies in patients with autoimmune encephalitis, removal of antibodies using immunoadsorption accelerates recovery in patients with antibodies against leucine-rich, glioma inactivated 1 (LG1), contactin-associated protein-2 (CASPR2) or NMDAR (Dogan Onugoren et al., 2016). Similarly, plasma exchange resulted in marked improvement in seizure frequency in a patient with anti-GAD antibody-related epilepsy in conjunction with substantial reduction of autoantibody burden (Farooqi et al., 2015). Substantiating a specific role for B cells, B cell depletion with rituximab has reported efficacy in refractory autoimmune encephalitis (Lee et al., 2016b; Strippel et al., 2017). Furthermore, plasma cell depletion with the proteasome inhibitor bortezomib has been reported to be effective in a case of extremely severe refractory anti- NMDAR encephalitis (Sveinsson et al., 2017).

Autoimmune hepatitis (AIH)

Autoimmune hepatitis is an immune-mediated liver disorder characterised by autoantibodies, elevated IgG levels and hepatitis. AIH is associated with a striking plasma cell infiltration/ accumulation in lobular and periportal hepatic regions as a hallmark feature present in "'90-100% of cases, including with acute presentations (Fujiwara et al., 2008; Nguyen Canh et al., 2017). Flow cytometry analysis of peripheral blood of patients with new-onset Al H indicates an expansion in circulating B cells, activated B cells and plasma cells compared to controls. Notably such Al H patients also exhibit an increase in circulating T follicular helper cells, key regulators of humoral immunity through their promotion of the germinal centre response (Ma et al., 2014). Moreover, significantly increased serum IL-21 - a key cytokine produced by T follicular helper cells which acts to promote B cell differentiation in antibody-secreting cells (Bryant et al., 2007) - is present in patients with AIH compared to healthy controls and positively correlates with serum levels of IgG, IgA and IgM (Ma et al., 2014).

Al H is associated with characteristic autoantibodies, with type 1 AIH exhibiting anti-nuclear (ANA) and/or anti-smooth muscle (SMA) autoantibodies, while type 2 Al H features anti-liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibodies (Liberal et al., 2013). Notably anti-ANA and SMA titres reduce or disappear with effective therapy in type 1 AIH (Liberal et al., 2013). While the precise pathogenic role of these autoantibodies is debated, the frequency of detection of these antibodies, prominence of plasma cells histologically and correlation of serum IgG and autoantibody levels/type (including anti-liver specific) with disease activity (including histology, aminotransferase levels and disease severity) strongly support a humoral/antibody-mediated component to AIH (Jensen et al., 1978; Ma et al., 2002; Sebode et al., 2018). Furthermore, isolated hepatocytes from patients with AIH are covered with surface immunoglobulin of the IgG subclass which is associated with greater susceptibility to antibody-dependent cell-mediated cytotoxicity in vitro (Vergani et al 1987).

Supporting a role for B cells in AIH, serum levels of BAFF are elevated in Al H, positively correlate with markers of liver injury and dysfunction (aminotransferases and bilirubin) and fall in response to corticosteroid treatment (Migita et al., 2007). B cell depletion with anti-CD20 antibody dramatically reduces liver inflammation in and alanine aminotransferase levels in a mouse model of Al H (Beland et al., 2015). Similarly, patients with treatment refractory AIH have been shown to respond to B cell depletion using the anti-CD20 monoclonal antibody, rituximab (Burak et al., 2013).

Chronic autoimmune urticaria (chronic spontaneous urticaria, CSU)

Chronic autoimmune urticaria or chronic idiopathic urticaria, now termed chronic spontaneous urticaria (CSU) is a skin disorder associated with mast cell and basophil degranulation with associated release of histamine, leukotrienes, prostaglandins and other substances resulting in recurrent weals (hives), angio-oedema or both for over 6 weeks (de Montjoye et al., 2018; Kolkhir et al., 2017). Activation of these cells is thought to be autoimmune mediated involving either a type I or type II hypersensitivity response, the latter referring to autoantibodies binding to antigens on target cells. IgG autoantibodies to IgE and FceRI (the high affinity receptor for IgE present on mast cells and basophils) are well-described in patients with CSU and in the case of the latter can promote receptor cross-linking and histamine release (Fiebiger et al., 1995; Hide et al., 1993; Sabroe et al., 2002; Sun et al., 2014; Tong et al., 1997).

Supporting a pathogenic functional role for these IgG autoantibodies (including anti-FceRI) is the finding that they can induce histamine release from healthy skin mast cells and basophils (Grattan et al., 1991; Niimi et al., 1996). In addition, IgG antibody can promote complement activation following cross-linking of FceRI to generate C5a which further enhances target cell degranulation (e.g.

basophil) and histamine release (Kikuchi and Kaplan, 2002). In addition, both heterologous and autologous injection of IgG anti- FceRI containing serum result in a weal and flare response (Kolkhir et al., 2017). Notably patients with positive autologous serum skin tests exhibit greater clinical severity (Caproni et al., 2004), longer duration and higher requirement for antihistamines (Staubach et al., 2006).

Removal of pathogenic autoantibodies using plasmapheresis has induced marked clinical responses in patients with severe unremitting CSU, in parallel with reduction in both serum IgG and in vitro measure of histamine-releasing activity (of patient serum on mixed leucocytes of healthy donors) (Grattan et al., 1992). Notably, the efficacy of omalizumab - a monoclonal antibody which selectively binds human IgE - is thought to in part be mediated through downregulation of FceRI density on mast cells and basophils (MacGlashan et al., 1997; Saini et al., 1999) thereby preventing IgG autoantibody-mediated cross-linking of adjacent receptors (Kaplan et al., 2017).

The source of these functional IgG autoantibodies is thought to be peripheral B cells (Chakravarty et al., 2011). CSU has been shown to be associated with polyclonal B cell activation, including production of other autoantibodies and increased serum IgE levels, together with enhanced B cell proliferation (Kessel et al., 2010; Toubi et al., 2000). Furthermore, serum levels of BAFF, a crucial B cell survival, activation and maturation signal, are elevated in patients with CSU and associate with disease severity (Kessel et al., 2012). B cell depletion using rituximab has reported remarkable clinical efficacy in refractory CSU and is associated with negative basophil histamine release assay (Chakravarty et al., 2011; Combalia et al., 2018; Steinweg and Gaspari, 2015).

Linear IgA disease (LAD)

Linear IgA disease (LAD) is a chronic, acquired, autoimmune subepidermal bullous skin disease characterised by IgA autoantibody deposition at the dermal-epidermal junction and/or by circulating IgA autoantibodies directed against heterogeneous basement membrane zone antigens

(Kasperkiewicz et al., 2010; Kirtschig and Wojnarowska, 1999; Utsunomiya et al., 2017).

Supporting a pathogenic role for IgA autoantibodies in LAD, immunoadsorption using a tryptophan- based immunoadsorper resulted in striking clinical improvement in LAD together with a reduction in total IgA (Kasperkiewicz et al., 2010). Supporting this are pre-clinical experiments demonstrating that passive transfer of IgA mouse monoclonal antibodies against a linear IgA antigen to SCID mice with human skin grafts can result in consistent IgA deposition at the basement membrane zone, neutrophil infiltration and basement membrane zone vesiculation (Zone et al., 2004).

Highlighting a role for B cells in LAD, B cell depletion with rituximab has evidenced clinical efficacy in severe/recalcitrant cases (Pinard et al., 2019).

IgA nephropathy

In IgA nephropathy, increased presence of poorly O-galactosylated IgAl glycoforms in the serum, subsequent O-glycan specific IgA and IgG autoantibody production (Suzuki et al., 2009) and resultant formation and deposition of IgAl immune complex in the glomerular mesangium serve to initiate renal injury and glomerulonephritis which can progress to renal failure (Lai et al., 2016; Tomana et al., 1999). Thus IgA or IgA immune complex deposition are regarded as fundamental causal factors in IgA nephropathy (Suzuki and Tomino, 2008). Serum levels of IgG and IgA autoantibodies (recognising galactose-deficient IgAl as an autoantigen) are significantly associated with progression of IgA nephropathy (dialysis/death) (Berthoux et al., 2012). Notably the serum concentration of autoantigen (galactose-deficient-lgAl) and IgG autoantibody correlate (Placzek et al., 2018). As a corollary, serum levels of galactose-deficient IgAl (autoantigen) driving pathogenic autoantibody production in IgA nephropathy independently associate with higher risk of deterioration in renal function (Zhao et al., 2012).

Further evidence supporting the importance of autoantibodies and the targeting of the specific cells producing these rather than generalised B cell depletion comes from a trial investigating the therapeutic potential of rituximab in IgA nephropathy (Lafayette et al., 2017). B cell depletion using rituximab in patients with IgA nephropathy with significant proteinuria and renal impairment failed to impact on serum levels of galactose-deficient IgAl and anti-galactose-deficient IgAl antibodies and, accordingly, did not favourably affect renal function (Lafayette et al., 2017).

Patients with IgA nephropathy have an expansion in bone marrow IgA plasma cells compared to controls, particularly subclass IgAl, suggesting that the bone marrow is the primary site of production of IgA deposited in the kidney mesangium in IgA nephropathy (van den Wall Bake et al., 1988). Furthermore, a positive correlation between bone marrow IgA plasma cells and serum IgA has been identified (Harper et al., 1994). Supporting these findings suggesting mephritogenic IgAl production in bone marrow, bone marrow transplantation has been reported to result in complete remission of IgA nephropathy (Iwata et al., 2006).

Patients with IgA nephropathy also feature a higher frequency of circulating memory B cells, activated B cells, T follicular helper cells and plasma cells (Sun et al., 2015; Wang et al., 2014b). Notably higher circulating levels of memory and activated B cells and T follicular helper cells correlated with more advanced disease (judged by proteinuria) (Sun et al., 2015). Higher serum levels of APRIL (a proliferation-inducing ligand, also known as TNFSF13), which mediates class switching largely for IgA and is critical for survival of bone marrow and mucosal plasma cells, associate with worse prognosis of IgA nephropathy (Han et al., 2016). A role for APRIL in genetic susceptibility to IgA nephropathy is also supported by genome-wide association studies (Yu et al., 2011). Furthermore, a role for aberrant expression of APRIL in tonsillar germinal centre B cells in IgA nephropathy has been found, correlating with greater proteinuria and suggesting a role for tonsillar B cells underlying the response of IgA nephropathy to tonsillectomy (Muto et al., 2017).

Vitiligo Vitiligo is an acquired chronic depigmenting disease resulting from selective melanocyte destruction (Ezzedine et al 2015).

Patients with vitiligo frequently exhibit autoantibodies at levels higher than controls, including anti- thyroperoxidase, anti-thyroglobulin, antinuclear, anti-gastric parietal cell and anti-adrenal antibodies (Liu and Huang, 2018), some of which correlate with clinical vitiligo activity (Colucci et al., 2014). In comparison to controls, vitiligo is associated with elevated total IgG, IgGl and lgG2 and melanocyte- reactive antibodies (Li et al., 2016b). The latter are most frequently directed against pigment cell antigens (Cui et al., 1992), including melanin-concentrating hormone receptor 1 (Kemp et al., 2002). Melanocyte death in vitiligo has been proposed to reflect apoptosis and is promoted in vitro by serum IgG from vitiligo patients (Ruiz-Arguelles et al., 2007). Notably IgG (and C3) deposits have been observed in the basement membrane zone of lesional skin. Furthermore, binding of IgG from vitiligo patients to cultured melanocytes increases with disease extent and activity, with further correlation of vitiligo activity to levels of anti-melanocyte IgA (Kemp et al., 2007) .

While there is debate regarded whether the presence of autoantibodies in vitligo reflects a primary cause or consequence of the disease, it is clear that vitiligo autoantibodies possess the capacity to result in pigment cell injury via multiple effector mechanisms, including antibody-dependent cellular cytotoxicity and complement-mediated cell damage in vitro (Cui et al., 1993; Norris et al., 1988).

MCHR function-blocking autoantibodies have also been identified in vitiligo patients, which would be expected to interfere with normal melanocyte function (Gottumukkala et al., 2006). In addition to the role of MCHR1 as a B cell autoantigen, the importance of B cells is further suggested in vitiligo through identification of Bcl-2 positive infiltrates in close juxtaposition to areas of depigmentation (Ruiz-Arguelles et al., 2007). Vitiligo has also been reported to respond to B cell depletion with monoclonal antibody to CD20 (Ruiz-Arguelles et al., 2013).

Notably T regulatory cells (Tregs) are deficient in vitiligo, together with an increase in PD-1 expressing Tregs suggesting Treg exhaustion and a possible role in the pathogenesis of vitiligo (Tembhre et al., 2015). This loss of suppression correlates with hyperactivation of CD8 ⁺ cytolytic T cells which are known to play a key role in vitiligo-induced depigmentation (Lili et al., 2012).

Primary biliary cirrhosis (PBC)

Primary biliary cirrhosis (PBC), also known as primary biliary cholangitis, is a chronic cholestatic liver disorder characterised pathologically by progressive small intrahepatic bile duct destruction with associated portal inflammation, fibrosis and risk of progression to cirrhosis, and serologically (>95%) by anti-mitochondrial antibody (AMA) and often an elevated serum IgM (Carey et al., 2015). Notably, autoantibodies (e.g. anti-centromere) are strongly associated with risk of progression to cirrhosis and portal hypertension (Nakamura, 2014).

While T cells have been reported to constitute the majority of cellular infiltrate in early PBC, B cells/plasma cells are also identified (Tsuneyama et al 2017). Specifically, formation of follicle-like aggregations of plasma cells expressing IgG and IgM around intrahepatic ducts have been noted in patients with PBC, further correlating with higher titres of AMA (Takahashi et al., 2012). The finding of oligoclonal B cell proliferation and accumulation of somatic mutations in liver portal areas from patients with PBC is consistent with antigen-driven B cell responses (Sugimura et al., 2003). A sustained rigorous B cell response in PBC has also been suggested through the finding of high levels of autoantigen-specific peripheral plasmablasts (to the pyruvate dehydrogenase complex autoantigen PDC-E2) consistent with ongoing activation of autoreactive B cells (Zhang et al., 2014). Notably, newly diagnosed patients with PBC exhibit elevated numbers of circulating T follicular helper cells and plasma cells, with both correlating positively with each other, as well as with levels of serum AMA and IgM (Wang et al., 2015). Rituximab has been reported to reduce serum total IgG, IgA and IgM, in addition to AMA IgA and IgM in patients with PBC and an incomplete response to ursodeoxycholic acid (Tsuda et al., 2012), in addition to a limited but discernible favourable effect on alkaline phosphatase and pruritus (Myers et al., 2013).

Primary sclerosing cholangitis (PSC)

PSC is a chronic liver disorder characterised by multifocal biliary strictures and high risk of cholangiocarcinoma, together with strong association with inflammatory bowel disease (Karlsen et al., 2017). A large number of autoantibodies have been detected in patients with PSC, but generally of low specificity, including pANCA, ANA, SMA and anti-biliary epithelial cell (Hov et al., 2008).

Notably and consistent with the known physiologically dominant role for secreted IgA in bile, the presence of autoreactive IgA against biliary epithelial cells correlates with faster clinical progression of PSC (to death/liver transplantation) (Berglin et al., 2013).

Functional IgA, IgM and IgG antibody secreting cells have been identified in PSC liver explants (Chung et al., 2016). Notably, the majority of these cells are plasmablasts rather than plasma cells (Chung et al., 2017). Alterations in the peripheral circulating T follicular helper cell compartment, a key facilitator of antibody responses, have been identified in PSC (Adam et al., 2018). Supporting a role for shared liver and gut adaptive immune response in PSC associated with inflammatory bowel disease, B cells of common clonal origin have been identified in both tissues together with evidence of higher somatic hypermutation consistent with (same) antigen-driven activation (Chung et al., 2018). As with PBC, a contribution from T follicular helper (T _F H) cells to disease pathogenesis is suggested by the presence of potentially pathogenic T _F H cells (CCR7 ^lo CXCR5 ⁺ PD-l ⁺ CD4 ⁺ T cells) (Adam et al., 2018). Notably genetic and functional data also support a role for impaired Foxp3 ⁺ regulatory T cell (Treg) function in contributing to the immune dysregulation of PSC (Sebode et al., 2014).

Notably PSC is also considered part of the spectrum of lgG4-related diseases (Gidwaney et al., 2017), a multiorgan fibroinflammatory disorder which is also associated with autoimmune pancreatitis and a robust elevation in circulating plasmablasts/plasma cells which reduce following treatment with glucocorticoids (Lin et al., 2017). This is associated with both an increase in class-switched memory B cells and T _F H cells, with IgG levels correlating to both circulating plasmablast and T _F H frequency and evidence of a marked tissue T _F H cell infiltration (Kubo et al., 2018). Substantiating the role of B cells in lgG4-related disease, B cell depletion with rituximab is effective in both induction and treatment of relapses (Ebbo et al., 2017).

Autoimmune thrombocytopenic purpura (immune thrombocytopenia; adult immune

thrombocytopenia)

Immune thrombocytopenia (ITP) is a disorder characterised by acquired thrombocytopenia (low platelet count) driven by immune recognition of platelet autoantigens and ensuing destruction of platelets.

Highlighting the importance of humoral immune mechanisms were early studies revealing that infusion of serum from patients with ITP to healthy volunteers resulted in profound

thrombocytopenia, that this was dose-dependent, that the humoral factor could be adsorbed by platelets and in the IgG fraction (Harrington et al., 1951; Karpatkin and Siskind, 1969; Shulman et al., 1965). In addition to IgG autoantibodies against platelet glycoprotein (GP) llb/llla, IgA and IgM anti platelet autoantibodies have been identified (He et al., 1994), as well as against other platelet surface proteins such as GPIb/IX, with a high degree of specificity for ITP (McMillan et al., 2003). These autoantibodies result in antibody-dependent platelet phagocytosis seen in vitro (Tsubakio et al., 1983) and in vivo by splenic macrophages and peripheral neutrophils (Firkin et al., 1969; Handin and Stossel, 1974). Notably the amount of platelet-associated IgG inversely correlates with the platelet count (Tsubakio et al., 1983).

In addition to promoting platelet destruction, autoantibodies have also been demonstrated to directly affect bone marrow megakaryocyte maturation (Nugent et al., 2009). Both GPIIb/llla and GPIb/IX are expressed on megakaryocytes, with autoantibodies found binding to these in ITP (McMillan et al., 1978). Furthermore, plasma from patients with ITP suppresses megakaryocyte production and maturation in vitro, an effect ameliorated through adsorption of autoantibody with immobilised antigen and also seen with patient IgG but not control IgG (McMillan et al 2004).

Splenectomy samples from patients with ITP show marked follicular hyperplasia with germinal centre formation and increased plasma cells consistent with an ongoing active B cell response in ITP (Audia et al 2011). Notably, frequency of splenic T _F H cells is higher in ITP compared to controls, with further expansions in splenic pre-germinal centre B cell, germinal centre B cell (in addition to plasma cells) also identified, and all correlating positively with percentage of T follicular helper cells (Audia et al., 2014). B cell depletion with rituximab is effective in improving platelet count in ~60% of patients with ITP, with patients in whom autoantibody is persistent more frequently failing to demonstrate a clinical response (Arnold et al., 2017; Khellaf et al., 2014). Highlighting an important role for long-lived plasma cells as a substrate for ongoing generation of pathogenic autoantibodies mediating platelet destruction and reduced production, patients who are refractory to B cell depletion with rituximab display autoreactive anti-Gpllb/llla plasma cells in spleen expressing a long- lived genetic programme (Mahevas et al., 2013).

T cells make an important contribution to the pathogenesis of ITP, with evidence of prolonged survival of autoreactive T cells and deficient Treg function (Wei and Hou, 2016).

Autoimmune Addison's disease (AAD)

AAD is a rare autoimmune endocrinopathy characterised by an aberrant immune destructive response against adrenal cortical steroid producing cells (Mitchell and Pearce, 2012).

A major autoantigen in AAD is steroid 21-hydroxylase with the majority (>80%) of patients exhibiting autoantibodies against this (Dalin et al., 2017), with sera from patients with AAD reacting with the zona glomerulosa of the adrenal cortex (Winqvist et al., 1992). Anti-adrenal antibodies are predictive of progression to overt disease or subclinical adrenal insufficiency in patients with other

autoimmune disorders (Betterle et al., 1997). Notably, levels of adrenal autoantibodies correlate with severity of adrenal dysfunction, suggesting association with the destructive phase of autoimmune adrenalitis. Conversely, patients exhibiting biochemical remission of adrenal dysfunction, including in response to corticosteroid therapy, also display loss of adrenal cortex autoantibody and 21-hydroxylase autoantibody (De Beilis et al., 2001; Laureti et al., 1998). While it is unclear whether these autoantibodies are directly pathogenic (particularly given their intracellular target), organ-specific reactive antibodies have been demonstrated from AAD sera (Khoury et al., 1981). Histologically, AAD is characterised by a diffuse inflammatory infiltrate, including plasma cells (Bratland and Husebye, 2011).

Genetic support for an important role for B cells in the susceptibility to AAD has come from the identification of BACH2 as a major risk locus (Eriksson et al., 2016; Pazderska et al., 2016). BACH2 encodes a transcriptional repressor which is required for class switch recombination and somatic hypermutation in B cells through regulation of the B cell gene regulatory network (Muto et al., 2010; Muto et al., 2004). Administration of rituximab to induce B cell depletion in AAD has reported efficacy in a new-onset case, with evidence of sustained improvement in cortisol and aldosterone (Pearce et al., 2012).

Supporting a T cell component to the pathogenesis of AAD, a high frequency of 21-hydroxylase- specific T cells is identifiable in patients, with CD8 ⁺ T cells able to lyse 21-hydroxylase positive target cells (Dawoodji et al., 2014).

Multiple sclerosis (MS)

MS is an inflammatory demyelinating disorder of the central nervous system (CNS).

While MS is typically conceptualised as a CD4 Thl/Thl7 T cell-mediated disorder, largely based on findings using the experimental autoimmune encephalomyelitis (EAE) model, T cell-specific therapies have not demonstrated clear efficacy in relapsing-remitting MS (Baker et al., 2017). In contrast, many active MS immunomodulatory and disease-modifying therapies are recognised to affect the B cell compartment and/or serve to deplete memory B cells, either physically or functionally (Baker et al., 2017; Longbrake and Cross, 2016).

The most well-recognised and persistent immunodiagnostic abnormality in MS - the presence of oligoclonal bands in cerebrospinal fluid (CSF) typically of IgG isotype (but also IgM) - is a product of B lineage cells (Krumbholz et al., 2012). Notably clonal IgG in CSF is stable over time, consistent with local production from resident long-lived plasma cells or antibody secreting cells maturing from memory B cells (Eggers et al., 2017). That anti-CD20 therapy reduces CSF B cells with no significant impact on oligoclonal bands suggests a substantial role for long-lived plasma cells in oligoclonal band production (Cross et al., 2006). Correlation of immunoglobulin proteomes in CSF samples has revealed strong overlap with transcriptome of CSF B cells highlighting the latter as the source (Obermeier et al., 2008). The majority of B cells in the CSF of patients with MS are memory B cells and short-lived plasmablasts, with the latter representing the main source for intrathecal IgG synthesis and correlating with parenchymal inflammation revealed by MRI (Cepok et al., 2005), with evidence of greater involvement in acute inflammation associated with relapsing-remitting MS (Kuenz et al 2008).

Pathologically, organised ectopic tertiary lymph node-like structures with germinal centres are present in the cerebral meninges in MS (Serafini et al., 2004). As with parenchymal lesions, B cell clones in meningeal aggregates largely use IgG (~90%, remainder IgM) (Lovato et al., 2011).

Moreover, antigen experienced B cell clones are shared between these meningeal aggregates and corresponding parenchymal lesions (Lovato et al., 2011). In addition, flow cytometry with deep immune repertoire sequencing of peripheral blood and CSF B cells indicate that peripheral class- switched B cells, including memory B cells, have a connection to the CNS compartment (Palanichamy et al., 2014). Notably memory B cells have recently been demonstrated to promote autoproliferation of Thl brain-homing autoreactive CD4 ⁺ T cells in MS (Jelcic et al., 2018).

The best characterised autoantigen in MS is myelin oligodendrocyte glycoprotein (MOG), the target of autoantibodies in EAE and against which antibodies are identified in ~20% children but relatively few adults with demyelinating disorders (Krumbholz et al., 2012; Mayer and Meinl, 2012). Evidence supporting a role for pathogenic autoantibody in MS includes the efficacy of plasma exchange in some patients (Keegan et al., 2005) and the presence of complement-dependent

demyelinating/axopathic autoantibodies in a subset of patients with MS (Elliott et al., 2012). Other autoantibodies have been identified against axoglial proteins around the node of Ranvier including autoantibodies against contactin-2 and neurofascin, with evidence of axonal injury evident using in vivo models when transferred with MOG-specific encephalitogenic T cells and inhibition of axonal conduction when used with hippocampal slices in vitro (Mathey et al., 2007).

Substantiating a key role for B cells in relapsing-remitting MS, B cell depletion using the chimeric anti-CD20 antibody rituximab reduces both inflammatory brain lesions and clinical relapses (Hauser et al., 2008). Similar unequivocally positive efficacy findings have been observed with use of other CD20 depleting agents such as ocrelizumab (humanised monoclonal anti-CD20 antibody) in relapsing MS (Hauser et al., 2017) and primary progressive MS (Montalban et al., 2017).

Illustrating cross-talk between B cells and T cells in MS, circulating T _F H cells are expanded in MS, correlating with progression of disease, and also present in lesions where they can promote inflammatory B cell function including antibody secretion (Morita et al., 2011; Romme Christensen et al., 2013; Tzartos et al., 2011).

Type 1 diabetes mellitus (T1DM) T1DM is an autoimmune disorder characterised by immune-mediated destruction of the pancreatic islet b cells. While the major cellular effectors of islet b cell destruction are generally considered as islet antigen-reactive T cells, a large body of evidence implicates B cells in this process and the pathogenesis of the disease (Smith et al., 2017).

The non-obese diabetic (NOD) mouse model of autoimmune diabetes exhibits an autoimmune insulitis. B cell deficient NOD mice exhibit suppression of insulitis, preservation of islet b cell function and protection against diabetes compared to NOD mice, indicating that B cells are essential for the development of diabetes in this model (Akashi et al., 1997; Noorchashm et al., 1997). Similar findings have been observed through use of anti-CD20 mediated B cell depletion, including reversal of established hyperglycaemia in a significant proportion of mice (Hu et al., 2007). Substantiating an important role for B cells in the pathogenesis of human T1DM, B cell depletion using rituximab results in partial preservation of islet b cell function in patients with newly diagnosed T1DM at 1 year (Pescovitz et al., 2009).

Studies with NOD mice suggest that islet autoantigen presentation by B cells to T cells is an important component of their pathogenic effect (Marino et al., 2012; Serreze et al., 1998).

Alterations in peripheral blood B cell subsets have been identified in T1DM patients, including reduction in transitional B cells and an increase in plasmablast numbers (Parackova et al., 2017). In addition, circulating activated T follicular helper cells are increased in children with newly diagnosed T1DM and autoantibody positive at-risk children (Viisanen et al., 2017).

The preclinical phase of T1DM is characterised by the presence if circulating islet autoantibodies, such as glutamic acid decarboxylase 65 (GAD65) and insulinoma antigen 2 (IA2) autoantibodies. The majority of children genetically at risk for T1DM with multiple islet autoantibody serocoversion subsequently progress to clinical diabetes (Ziegler et al., 2013). While these autoantibodies are predictive of development of T1DM, their precise pathogenic role is debated. Supporting evidence for their pathogenicity comes from studies in NOD mice where elimination of maternal transmission of autoantibodies from prediabetic NOD mice protects progeny from development of diabetes (Greeley et al., 2002). Notably, NOD mice deficient in activating Fc receptors for IgG (FcyR) are protected from spontaneous onset of T1DM (Inoue et al., 2007).

Coeliac disease and dermatitis herpetiformis

Coeliac disease is a chronic immune-mediated enteropathy against dietary gluten in genetically predisposed individuals (Lindfors et al., 2019). Adaptive immune responses play a key role in the pathogenesis of coeliac disease characterised by both antibody production towards wheat gliadin (IgA and IgG) and tissue transglutaminsase 2 enzyme (TG2) (IgA isotype), together with gluten- specific CD4 ⁺ T cell responses in the small intestine (van de Wal et al., 1998). The finding of TG2 as the primary autoantigen present in endomysium and the target for endomysial antibodies secreted by specific B cells (Dieterich et al., 1997) forms the basis of the primary coeliac antibody test used to support a diagnosis of coeliac disease with ~ 90-100% sensitivity/specificity (Rostom et al., 2005).

Multiple potentially pathogenic effects have been ascribed to coeliac disease autoantibodies (Caja et al., 2011) including of the IgA subclass, such as: interference with intestinal epithelial cell differentiation (Halttunen and Maki, 1999); promotion of retrotranscytosis of gliadin peptides to enable their entry into the intestinal muscosa to trigger inflammation (Matysiak-Budnik et al., 2008); increased intestinal permeability and induction of monocyte activation (Zanoni et al., 2006); and inhibition of angiogenesis via targeting of blood vessel TG2 in the lamina propria (Myrsky et al., 2008).

B cells specific for gluten and TG2 have been proposed to act as antigen-presenting cells to gluten- specific CD4 ⁺ T cells, with HLA-deamidated gluten peptide-T cell receptor interaction resulting in activation of both T and B cell, the latter differentiating into plasma cells with ensuing production of antibodies targeting gliadin and endogenous TG2 (du Pre and Sollid, 2015; Sollid, 2017).

While genetic association studies highlight a key role for CD4 ⁺ T cells in the pathogenesis of coeliac disease, integrative systems biology approaches have highlighted a significant role for B cell responses in coeliac disease (with disease SNPs significantly enriched in B-cell-specific enhancers) (Kumar et al., 2015).

Patients with active coeliac disease exhibit a marked expansion of TG2-specific plasma cells within the duodenal mucosa. Further increases in extracellular IgM and IgA are evident in the lamina propria and epithelial cells in response to gluten, consistent with an active immunoglobulin response within the small intestinal mucosa (Lancaster-Smith et al., 1977). Notably TG2-specific IgM plasma cells have been described in coeliac disease, which could exert pathogenic effects via their ability to activate complement to promote inflammation. Indeed, subepithelial deposition of terminal complement complex has been observed in untreated and partially treated (but not successfully treated) patients with coeliac disease, correlating with serum levels of gluten-specific IgM and IgG (Halstensen et al., 1992).

Dermatitis herpetiformis is an itchy blistering skin disorder regarded as the cutaneous manifestation of coeliac disease (Collin et al., 2017). It is characterised by granular IgA deposits in the dermal papillae of uninvolved skin (Caja et al., 2011). Patients with dermatitis herpetiformis exhibit autoantibodies against epidermal TG3, which are gluten-dependent, and respond slowly to a gluten- free diet (Hull et al., 2008). Its pathogenesis is thought to involve active coeliac disease in the intestine resulting in the formation of IgA anti-TG3 antibody complexes in the skin.

Notably B cell depletion with rituximab has resulted in complete clinical and serological remission in a case of refractory dermatitis herpetiformis (Albers et al., 2017). Similarly, rituximab has resulted in dramatic clinical improvement in a mixed case of symptomatic coeliac disease and Sjogren's syndrome (Nikiphorou and Hall, 2014).

Psoriasis

Psoriasis is a chronic, immune-driven disease primarily affecting the skin and joints (Greb et al., 2016). Pathophysiologically, psoriasis involves components of innate and adaptive immunity, particularly involving T cell (specifically T _H 17 cell) signalling, dendritic cells and keratinocytes (Greb et al., 2016).

Analysis of psoriatic arthritis synovium has revealed frequent ectopic lymphoid neogenesis which can drive local antigen-driven B cell development, which notably regressed with treatment (Canete et al., 2007). Critically these tertiary lymphoid structures triggered by persistent inflammation contain highly organised follicles, segregated B cell and T cell zones and follicular dendritic cell networks providing the substrate for a germinal centre response to support local (aberrant) adaptive immune responses against locally displayed antigens, including autoreactive lymphocyte clone cell survival and pathogenic immunoglobulin production (Canete et al., 2007; Pipi et al., 2018).

Psoriasis has recently been identified to be associated with several serum autoantibodies, including IgG against LL37 (cathelicidin) and ADAMTSL5 (a disintegrin and metalloprotease domain containing thrombospondin type 1 motif-like 5), whose levels correlate with psoriasis clinical severity and reflect disease progression over time (Yuan et al., 2019). Notably expression of these autoantigens is reduced by effective therapy targeting IL-17 or TNF-a, suggesting positive regulation and feedforward induction by psoriasis disease-related pro-inflammatory cytokines (Fuentes-Duculan et al., 2017). Other autoantibodies identified such as those against anti-a6-integrin have been proposed to contribute to induction of a chronic wound healing phenotype (Gal et al., 2017).

Analysis of total circulating immunoglobulins in psoriasis has revealed elevated total IgA, but not total IgG or IgM (Kahlert et al., 2018). Supporting this increase, an elevation in plasmablast levels in psoriasis has also been noted (Kahlert et al., 2018).

Analysis of peripheral blood lymphocyte subsets has revealed an expansion in circulating activated B cells and T _FH cells together with elevated serum IL-21 in psoriasis compared to healthy donors; notably the levels of each of these correlated positively with psoriasis severity (Niu et al., 2015). Substantiating the functional importance of this, circulating T _F H cells from psoriasis patients exhibit signs of activation and produce higher levels of cytokines, with significant reduction in these on treatment. Moreover, psoriasis lesions exhibit extensive T _F H infiltration (Wang et al., 2016b). IL-10 producing regulatory B cells (i.e. B10 cells) have been found to be reduced in psoriasis, exhibit impaired activity and inversely correlate with IL-17 and IFN-y producing T cells (Mavropoulos et al., 2017).

There are reports of B cell depletion using rituximab inducing de novo psoriasis skin lesions (Dass et al., 2007), although this is debated (Thomas et al., 2012), but improved arthritis (Jimenez-Boj et al., 2012), highlighting the complex role of B cells in the pathogenesis of the disease and the importance of non-canonical B cell function (i.e. beyond autoantibody production) including but not limited to cytokine production and antigen presentation to influence autoreactive T cells (Hayashi et al., 2016; Yoshizaki et al., 2012).

The idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM) and polymyositis (PM)

DM and PM are inflammatory myopathies typically resulting in symmetrical proximal myopathy that differ in clinical features, pathology and clinical response/prognosis (Findlay et al., 2015). DM is characterised by skin lesions and (usually except in amyopathic cases) inflammation of skeletal muscle. PM is traditionally the term ascribed to idiopathic inflammatory myopathy which is neither DM nor sporadic inclusion body myositis (Findlay et al., 2015). Other subtypes of IIM recognised include necrotising autoimmune myositis and overlap syndrome (Dalakas, 2015).

Supporting a role for B cells, IIMs are associated with autoantibody production, both myositis- specific and myositis-associated, useful clinically in diagnosis, including for DM (Anti-MDA-5, anti-Mi- 2, anti-TIF-1, anti-NXP-2), PM (anti-synthetase antibodies), necrotising autoimmune myositis (anti- FIMGCR, anti-SRP) and inclusion body myositis (anti-cNIA) (Dalakas, 2015). Notably autoantibody levels in patients with myositis have been shown to reduce with B cell depletion and correlate with changes in disease activity (Aggarwal et al., 2016).

DM is thought to be substantially humorally mediated through pathogenic antibody-mediated complement activation on endothelial cells resulting in necrosis and ischaemia and muscle fibre destruction (Kissel et al., 1986), i.e. a complement-mediated microangiopathy. Indeed, ectopic lymphoid structures have been identified in skeletal muscle of patients with DM, including evidence of germinal centres with dark/light zone organisation and molecular evidence of in situ B cell differentiation (Radke et al., 2018). PM and inclusion body myositis have traditionally been regarded as primarily CD8 ⁺ cytotoxic T cell-mediated disorders, however abundant enrichment of plasma cells has been identified in muscle biopsies from patients with these disorders and associated high expression of immunoglobulin transcript (Greenberg et al., 2005). Further supporting a local B cell antigen-specific response in PM and inclusion body myositis is the finding of affinity maturation (encompassing somatic mutation, class switching and oligoclonal expansion) within IgH chain gene transcripts of local B cells and plasma cells in patients but not in control muscle tissue (Bradshaw et al., 2007). Similar B cell clonal diversification has been noted in DM consistent with an antigen- driven chronic B cell response in inflamed muscle (McIntyre et al., 2014).

Serum levels of BAFF (B cell-activating factor belonging to the tumour necrosis factor family), a critical factor in B cell survival and maturation, is significantly elevated in DM in association with increased expression of BAFF in the perifascicular area of skeletal muscle of patients versus normal controls (Baek et al., 2012). Notably expression of BAFF receptors have been co-localised to or in the vicinity of plasma cells and B cells in patients with myositis with a correlation between the number of cells expressing BAFF receptors and plasma cell frequency, particularly those expressing anti-Jo-1 or anti-Ro52/Ro60 autoantibodies, consistent with local BAFF-driven differentiation of plasma cells in myositis (Krystufkova et al., 2014). Supporting a functional role for these changes, BAFF pathway expression is positively correlated with measures of disease activity in idiopathic inflammatory myopathies (Lopez De Padilla et al., 2013).

Supporting a key pathogenic role for B cells in the idiopathic inflammatory myopathies, refractory skin rashes have shown improvement in response to B cell depletion using rituximab (Aggarwal et al., 2017), with evidence of some clinical response in patients with DM or PM (Mok et al., 2007; Oddis et al., 2013; Sultan et al., 2008).

Highlighting a specific role for T-B cell interaction and CD4 ⁺ T cell help for B cell responses in DM, alteration in circulating T _F H cell subsets have been observed skewed towards subtypes favouring B cell help to promote immunoglobulin production via IL-21 (Morita et al., 2011). Notably such circulating T _F H cells promote differentiation of naive B cells to plasmablasts (Morita et al., 2011).

Interstitial lung disease (ILD)

ILD encompass a complex and heterogeneous set of disorders, including idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis, drug-associated ILD, sarcoidosis and ILD associated with connective tissue disorders and familial/other syndromes (Wallis and Spinks, 2015). Supporting a role for B cells in driving the progression of ILD, use of rituximab in patients with severe, progressive non-IPF ILD refractory to conventional immunosuppression shows evidence of improvement in lung capacity and stabilisation of diffusing capacity of carbon monoxide (Keir et al 2012; Keir et al 2014). Striking clinical improvement has also been reported in response to rituximab in a case of severe refractory hypersensitivity pneumonitis (Lota et al., 2013), a condition associated with germinal cell formation in bronchus-associated lymphoid tissue (Suda et al., 1999). Favourable responses to B cell depletion have also been reported in severe cases of ILD associated with anti-synthetase (Sem et al., 2009) and systemic sclerosis (Sari et al., 2017).

IPF is associated with circulating IgG autoantibodies (Feghali-Bostwick et al., 2007), with

morphological evidence of microvascular injury in association with IgG, IgM and IgA deposition within septal microvasculature suggesting antibody-mediated microvascular injury (Magro et al., 2006). Autoantigens identified include annexin 1, with evidence of significant elevation in autoantibody targeting annexin 1 during acute exacerbations of IPF (Kurosu et al., 2008) suggesting a potential role in these episodes. Notably immune complex formation between antigens and immunoglobulin - a potent trigger of inflammation and secondary injury - are present in IPF in the circulation (Dobashi et al., 2000), lung parenchyma (with complement deposition) (Xue et al., 2013) and from bronchoalveolar lavage.

Histology of lungs of patients with IPF has also identified abnormal B cell aggregates including germinal centre formation, particularly close to fibroproliferative areas (Campbell et al., 1985; Marchal-Somme et al., 2006). Moreover, IPF is associated with elevated circulating and local CXCR13 - a CD4 ⁺ T cell-derived chemokine promoting pathological B cell trafficking and formation of ectopic lymphoid-like structures and elevated in several autoantibody-mediated disorders - and this elevation correlates with exacerbations and poor outcomes suggesting a pathogenic role for CXCR13 and B cells in IPF (Vuga et al., 2014; Yoshitomi et al., 2018). Moreover, the circulating plasmablast pool is expanded in IPF, with evidence of greater antigen differentiation of circulating B cells and significantly increased plasma levels of BLyS (B lymphocyte stimulating factor) a key promoter of B cell survival and differentiation, with patients displaying the highest levels of BLyS also those with the lowest 1-year survival rates (Xue et al., 2013).

In the setting of IPF, evidence exists supporting a role for targeting pathogenic autoantibody using therapeutic plasma exchange and rituximab to alleviate acute respiratory exacerbations in critically ill patients with IPF which can otherwise be fatal within days (Donahoe et al., 2015). Notably plasma exchange was associated with a reduction in anti-Hep-2 autoantibodies in patients responding to treatment (Donahoe et al., 2015). Inflammatory bowel disease (IBD) - ulcerative colitis (UC) and Crohn's disease (CD)

UC is an idiopathic IBD characterised by inflammation of the colon and rectum.

UC is associated with an expanded circulating plasmablast subset of B cells together with elevated serum IgG (Wang et al 2016a). Notably, inflammatory markers (CRP and ESR) correlate positively with levels of plasmablasts and serum IgG levels. Conversely, treatment with mesalazine lowers plasmablast levels in UC (Wang et al., 2016a).

UC is associated with autoantibody formation mainly antineutrophil cytoplasmic antibodies (ANCA) and anti-goblet cell antibodies with the latter considered potentially specific and both aiding differentiation from CD in early cases (Conrad et al., 2014). Underlining a pathogenic role for autoantibodies in UC is the finding of complement activation in relation to epithelial-bound IgG (Brandtzaeg et al., 2006). The known substantial infiltration of the colon with B cells and plasma cells in UC, as in CD, provides a local source for these (Cupi et al., 2014).

Highlighting a role for altered T follicular regulatory and T _FH subsets, key T cell subsets whose balance regulates B cell responses, patients with UC exhibit an increase in circulating T _FH cells but lower T follicular regulatory cell levels, in conjunction with elevated IL-21 and reduced IL-10 (Wang et al., 2017). Notably, serum IL-21 level and circulating T _FH cell level positively correlate with clinical severity score and systemic inflammatory markers, with the converse holding for levels of circulating T follicular regulatory (T _FR ) cells and IL-10 (Wang et al., 2017). This imbalance in the T _FR /T _FH ratio has been observed also in other canonical B cell driven pathogenic immunoglobulin-mediated disorders such as myasthenia gravis.

While B cell depletion with rituximab has not proven effective in steroid-unresponsive moderate UC in a clinical trial setting (Leiper et al., 2011), colon-resident plasma cells have been shown to be unaffected by this therapy, suggesting failure to target this B cell cellular/anatomic compartment may contribute to the observed lack of efficacy (Uzzan et al., 2018). Notably the pathogenic effects of plasma cells may not be limited to pathogenic autoantibody production - both UC and CD are characterised by mucosal accumulation of lgA ⁺ plasma cells expressing granzyme B, a serine protease induced by IL-21 in B cells and linked to induction of apoptosis after cytotoxic cellular attack (Cupi et al., 2014; Hagn et al., 2010).

CD is characterised by transmural inflammation of the gastrointestinal tract and affects any part of it and, like UC, exhibits a significant increase in plasma cells in the intestinal lamina propria as a source of both IgG and monomeric IgA (Uzzan et al., 2018). Notably, IgG plasma cells correlate with the severity of intestinal inflammation (Buckner et al., 2014). Furthermore, B cells are seen to localise around a key pathological hallmark of CD, intestinal granulomas (Timmermans et al 2016). Analysis of circulating class switched memory B cells in CD reveals increased levels of somatic hypermutation consistent with chronic stimulation (Timmermans et al., 2016). Notably, alterations in the peripheral B cell compartment improve with effective treatment of inflammation through targeting of TNF-a (Timmermans et al., 2016).

As with UC, patients with CD show abnormal B cell responses in the form of detectable (IgG/lgA) auto- or anti-microbial antibodies, including against Saccharomyces cerevisiae antibodies (ASCA) and neutrophils (ANCA), with serological markers predictive of disease prior to diagnosis (Quinton et al., 1998; van Schaik et al., 2013), as well as of risk of recurrence post-surgical resection (Hamilton et al., 2017). Underlining the pathogenic potential of these, autoantibodies against the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) are produced by lamina propria cells and have been associated with stricturing behaviour, which may reflect their ability to reduce neutrophil function, and increased intestinal permeability (Jurickova et al., 2013).

Highlighting a role for T cells contributing to the observed B cell phenotype of CD, circulating T _FH cells are increased in patients with CD versus controls (Wang et al., 2014c).

Autoimmune uveitis and autoimmune retinopathy

Uveitis refers to inflammation of the tissues of the eye, ranging from the anterior chamber which includes the iris and ciliary body, to the vitreous, to posterior structures (retina or choroid) (Smith et al., 2016). Notably uveitis is observed in association with systemic autoimmune and inflammatory diseases, such as seronegative spondyloarthritis, IBD, psoriatic arthropathy, Behcet's disease, rheumatoid arthritis, juvenile idiopathic arthritis, in addition to infectious and other aetiologies (Selmi, 2014). Autoimmune uveitis is therefore a collection of disorders in which there is loss of ocular immune privilege and which can be associated with disease affecting other tissues.

Autoimmune retinopathy is associated with progressive loss of visual acuity in association with anti- retinal antibodies (Grange et al., 2014). Autoantibodies against multiple retinal proteins have been identified, including retinal specific proteins such as recoverin localised in photoreceptors and a- enolase (Ren and Adamus, 2004), the former also described in cancer-associated retinopathy. Anti- recoverin antibodies are able to penetrate retinal layers to promote apoptotic photoreceptor cell death (Adamus, 2003). Notably patients with autoimmune retinopathy exhibit altered peripheral mature B cell memory subsets, including evidence of activation of naive memory B cells and altered isotype profile (Stansky et al., 2017). Murine models of autoimmune uveitis suggest T helper cells, specifically THI and TH17 cells as being important effectors. However, B cells are felt to play in important pathogenic role through uveal antigen presentation and subsequent activation of T cells (Prete et al., 2016), inflammatory cytokine production and support of T cell survival (Smith et al., 2016). Antigens involved are thought to include melanocyte components or tyrosinase or related proteins including recoverin, rhodopsin and retinal arrestin (Prete et al., 2016). In addition to direct cell toxicity described above for retinal autoantibodies, autoantibodies in autoimmune uveitis may exert pathogenic effects through formation of antigen-antibody immune complexes to trigger innate immune mechanisms or complement activation via the classical pathway (Smith et al., 2016). As a corollary, mice deficient in complement (C3) develop less severe experimental autoimmune uveitis than controls (Read et al., 2006).

Evidence for involvement of B cells in autoimmune uveitis include: the presence of B cells in the intra-ocular inflammatory infiltrate and vitreous immunoglobulin (Godfrey et al., 1981; Nguyen et al., 2001), remission of ocular disease in association with onset of combined variable

immunodeficiency (CVID, a primary immunodeficiency syndrome associated with impaired B cell differentiation and hypogammaglobulinaemia) (Amer et al., 2007), elevation of serum BAFF in autoimmune disease with co-existing uveitis (Gheita et al., 2012) and the response to rituximab (described below).

Highlighting a role for B cell mediated homeostatic regulation of T cell function that is perturbed in an experimental model of uveitis, tonic inhibition of T cell trafficking by B cell derived peptide release (PEPITEM) is lost, facilitating T cell recruitment to promote chronic tissue injury (Chimen et al., 2015). Furthermore, IL-35 promoted induction of regulatory B cells is protective in experimental autoimmune uveitis, in part through inhibition of pathogenic TH17 and THI cells whilst enhancing expansion of Treg cells (Wang et al., 2014a).

Notably, B cell depletion with rituximab has shown efficacy in stabilising and/or improving visual acuity in patients with autoimmune retinopathy (Maleki et al., 2017) and autoimmune uveitis and scleritis (Hardy et al., 2017; Pelegrin et al., 2014).

Mixed connective tissue disease (MCTD) and undifferentiated connective tissue disease (UCTD)

MCTD is a systemic autoimmune disorder characterised by the presence of antibodies to Ul-RNP (Ul-ribonuclear protein).

In addition to acting as a serological hallmark for MCTD diagnosis, anti-Ul RNP autoantibodies are thought to play a central pathogenic role (Tani et al., 2014), including binding to pulmonary artery endothelial cells (that may promote pulmonary hypertension via triggering of endothelial cell inflammation) (Okawa-Takatsuji et al., 2001). Further evidence strongly suggesting a role for this antibody in the pathogenesis of MCTD comes from studies involving immunisation of mice with antigenic peptide of the Ul-70-kd subunit of the U1 snRNP in which induction of anti-RNP antibodies and MCTD-like autoimmunity including interstitial lung disease resulted (Greidinger et al., 2006). Autoantibodies are also thought to promote tissue injury in MCTD via immune complex formation and complement activation (Szodoray et al., 2012).

Beyond Ul-RNP, other findings highlighting altered humoral adaptive immunity in MCTD are the frequent presence of other autoantibodies (e.g. ANA), hypergammaglobulinaemia and polyclonal B cell hyperreactivity and activation (Hajas et al., 2013).

Consistent with altered B cell homeostasis in MCTD, analysis of peripheral B cell subsets reveals altered numbers of transitional cells, naive B cells and memory B cells, together with increased plasma cell number correlating with levels of anti-Ul-RNP (Hajas et al., 2013). Furthermore, in common with other connective tissue disorders, abnormalities of bone marrow are reported including increase in plasma cell number in association with lymphoid aggregates (Rosenthal and Farhi, 1989).

Supporting an important role for B cells in the pathology of MCTD, B cell depletion using rituximab has been shown to stabilise pulmonary function in patients with associated interstitial lung disease (Lepri et al., 2016). Further supporting a role for pathogenic immunoglobulin and/or immune complexes in MCTD, plasmapheresis (Seguchi et al., 2000), immunoadsorption (Rummler et al.,

2008) including combined with anti-CD20 therapy (Rech et al., 2006) has reported efficacy.

Highlighting a T cell component likely to contribute to the pathogenesis of MCTD, levels of circulating Tregs are reduced and even lower in patients with active disease.

UCTD describes a group of unclassifiable systemic autoimmune diseases which overlap with serological and clinical features of definite connective tissue diseases (CTD), e.g. SLE, systemic sclerosis, DM, PM, MCTD, rheumatoid arthritis and Sjogren's syndrome, but which do not fulfil criteria for classification into a specific CTD (Mosca et al., 2014). Notably a significant proportion of these patients go on to evolve into a defined CTD (Mosca et al., 2014). Patients often exhibit positive anti-nuclear antibodies (ANA).

Patients with UCTD have been shown to exhibit significantly increased expression of the activation marker CD86 on circulating B cells with nominal but non-statistically significant increases in circulating plasma cells and T _FH cells (Baglaenko et al., 2018). Highlighting a T cell component to the disease, patients with UCTD show lower levels of circulating CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells (Tregs) together with elevated INF-y production (Szodoray et al., 2008).

Autoimmune connective tissue disease such as systemic lupus erythematosus (SLE); discoid lupus erythematosus (DLE)

SLE is a multisystem archetypal autoimmune connective tissue disease (CTD) predominantly affecting women with a predilection for affecting the kidneys, joints, central nervous system and skin and the presence of autoantibodies against nucleic acids and nucleoproteins (Kaul et al., 2016).

SLE is associated with a number of autoantibodies, some of which antedate the clinical onset by several years, such as IgG/lgM antiphospholipid antibodies, antinuclear antibodies (ANA) and others (McClain et al., 2004). Additional antibody targets and disease associations include: Clq, dsDNA and Smith (Sm) in lupus nephritis, Ro (SSA, Sjogren syndrome-related antigen) and La (SSB) in secondary Sjogren syndrome and cutaneous lupus, Ul-RNP and Ro in interstitial lung disease, prothrombin and b2 glycoprotein 1 in antiphospholipid syndrome (Kaul et al., 2016). Many of these autoantibodies are regarded as pathogenic, largely through the formation of immune complexes and deposition, e.g. in renal glomeruli and skin, to induce immune activation via complement activation or via Fc receptors. Immune complexes can promote B cell and dendritic cell activation leading to cytokine production (e.g. IFN-a) (Means and Luster, 2005), in addition to activating neutrophils via FcyRIIA to promote reactive oxygen species (ROS) and chemokine release inducing tissue damage (Bonegio et al., 2019).

Beyond autoantibody production indicating a breakdown of self-tolerance in B cells, multiple lines of evidence implicate B cells as major contributors to the pathophysiology of SLE. Patients with active lupus exhibit defects in central and peripheral B cell tolerance, responsible for silencing/removing autoreactive B cells (e.g. deletion, anergy and receptor editing in the bone marrow), which would facilitate the survival and activation of autoreactive B cells (Jacobi et al., 2009; Yurasov et al., 2005).

B cell hyperactivity and plasmacytoid dendritic cell interaction together with RNA-containing immune complexes serves to promote further B cell expansion (Berggren et al., 2017).

A mouse model exhibiting SLE-like pathology spontaneously forms germinal centres with increased plasma cell number and lowered threshold for B cell activation and impaired elimination of autoreactive B cells (Kil et al., 2012). Lupus prone mice display expansion of antigen-activated marginal zone (MZ) B cells which migrate to lymphoid follicles to engage with CD4 ⁺ T cells to promote autoantibody production, consistent with a breach in follicular exclusion (Duan et al., 2008; Zhou et al., 2011). B cell-T cell interaction is a critical contributor to the pathogenesis of SLE, including via activation of autoreactive B cells by T cell subsets and promotion of high-affinity autoantibodies from germinal centres supported by T _F H cells. Murine models of lupus demonstrate abnormal T _F H expansion and dysregulated germinal centre reactions correlating with autoantibody level (Kim et al., 2015), driven in part through elevated IL-21 (Bubier et al., 2009) and ICOS-dependent (Mittereder et al., 2016) signalling released/mediated by T _F H cells. Similarly, findings from patients with SLE indicate increased levels of active T _F H cells correlating with autoantibody titre, severity of organ involvement by disease and plasma cell number with evidence of downregulation in response to corticosteroids (Feng et al., 2012; Simpson et al., 2010), Notably these circulating T _F H cells are phenotypically similar to those present in germinal centres, correlate with circulating plasmablast levels and promote B cell differentiation to IgG-secreting plasma cells in vitro (Zhang et al., 2015).

Further supporting a role for B cells as key mediators of disease in SLE are observations of clinical efficacy with B cell depletion using rituximab in refractory patients (laccarino et al., 2015), including lupus nephritis except in rapidly progressive crescentic cases (Davies et al., 2013) and

neuropsychiatric lupus (Tokunaga et al., 2007). Notably, more rapid memory B cell and plasmablast repopulation post-rituximab are associated with earlier disease relapse (Vital et al., 2011). Notably rituximab use in SLE is also associated with altered cytokine levels and T cell phenotypes beyond simple B cell depletion highlighting an effect on the latter as a likely contributor to its efficacy (Tamimoto et al., 2008). Supporting a pathogenic role for autoantibodies in lupus, autoantibody removal using immunoadsorption has provided clinical benefits in refractory disease (Kronbichler et al., 2016).

DLE, the most common form of chronic cutaneous SLE, has been associated with polyclonal B cell activation (Wangel et al., 1984), together with increased numbers of B cells in skin (Hussein et al., 2008) which can promote skin fibrosis via cytokine release, further enhanced by BAFF (Francois et al., 2013) and a predominance of T cells (Andrews et al., 1986). Notably abnormalities in circulating B cells in discoid lupus similar to that of SLE have been identified, including a correlation with clinical disease criteria (Kind et al., 1986; Wouters et al., 2004). Furthermore, B cell depletion using rituximab has proven effective for cutaneous manifestations of SLE (Hofmann et al., 2013) and DLE (Quelhas da Costa et al., 2018).

Immune-mediated inflammatory disease (IMID) such as Scleroderma (SS, systemic sclerosis), rheumatoid arthritis and Sjogren's disease

SS is an immune-mediated inflammatory disease typified by fibrosis of the skin and internal organs together with a vasculopathy (Denton and Khanna, 2017). SS is associated with autoantibody formation including anti-centromere, anti-Scl-70, anti-RNA polymerase III (and other ANA), with strong relation to disease presentation/internal organ involvement and outcome (Nihtyanova and Denton, 2010). Evidence of autoantibodies as pathogenic drivers of the complications of SS include documentation of functional autoantibodies targeting platelet-derived growth factor receptor (PDGFR) which promote PDGFR stimulation and collagen and alpha-smooth muscle actin expression to support a pro-fibrotic phenotypic transition of fibroblasts (Gunther et al., 2015). Other functional autoantibodies detected in SS include against those targeting Angiotensin II type 1 receptor (AT1R) and endothelin type A receptor (ETAR), promoting agonistic activity at these receptors and strongly predictive of severe SS complications and mortality (Becker et al., 2014; Riemekasten et al., 2011).

SS is associated with polyclonal B cell activation and increased serum IgG (Famularo et al., 1989). Notably circulating B cells from patients with SS overexpress CD19 consistent with heightened intrinsic B cell activation which is expected to promote autoantibody production (Tedder et al.,

2005). Increased activation markers are also seen specifically in the memory B cell pool in SS, with enhanced ability to produce IgG in vitro (Sato et al., 2004). Notably the diffuse cutaneous variant of SS has been associated with an expanded circulating class-switched memory B cell population (Simon et al., 2016). Further supporting an alteration in B cell homeostasis in SS is the finding of an elevation in serum levels of key cytokines and B cell factors involved in regulating B cell activation, survival or homing, including IL-6, BAFF and CXCL13 (Forestier et al., 2018). Notably BAFF is upregulated in affected skin of patients with SS, with increases in serum levels of BAFF correlating with new onset or exacerbation of organ involvement and conversely reduction in serum BAFF observed with skin lesion regression (Matsushita et al., 2006).

Pathologically, cutaneous lesions have been shown to include cellular infiltrates containing plasma cells (Fleischmajer et al., 1977). Furthermore, highlighting a role for T cell regulators of autoantibody production by B cells, T cells possessing a T _FH phenotype including expression of ICOS are seen to infiltrate cutaneous lesions of SS and correlate with both dermal fibrosis and disease status clinically (Taylor et al., 2018). As a corollary, anti-ICOS antibody or IL-21 neutralisation administered to a murine model of SS-GVFID (graft-versus-host-disease) reduces dermal inflammation and/or fibrosis (Taylor et al., 2018).

Clinically, B cell depletion using rituximab has exhibited a beneficial effect on pulmonary function (or stabilisation) and improvement of skin thickening in SS associated with interstitial lung disease (Daoussis et al., 2017; Jordan et al., 2015). Rheumatoid arthritis (RA)

RA is associated with a large number of autoantibodies, most well described being rheumatoid factors and anti-citrullinated protein antibodies (ACPA) but including others such as anti- carbamylated protein antibodies and anti-acetylated protein antibodies. As with SLE, the presence of these autoantibodies can antedate clinical expression by years and also associate with radiographic disease progression (Derksen et al., 2017).

ACPA antibodies include IgG, IgA and IgM and given the presence of citrullinated protein in synovial fluid from inflamed RA joints, suggests that ACPA could bind these (Derksen et al., 2017). The collagen-induced arthritis mouse model develops antibodies against both CM and cyclic citrullinated peptide early after immunisation, with administration of murine monoclonal antibodies against citrullinated fibrinogen enhancing arthritis and binding inflamed joint synovium (Kuhn et al., 2006). Notably, the Fab-domain of ACPAs display a high abundance of N-linked glycans which may alter its properties to promote specific effector functions to ACPA IgG, such as binding of immune cells (Hafkenscheid et al., 2017). Immune complexes containing ACPA and citrullinated fibrinogen can stimulate TNF production via binding of Fey receptors on macrophages (Clavel et al., 2008), including macrophages derived from synovial fluid of patients (Laurent et al., 2011). Complement activation through autoantibodies is also a likely mechanism of pathogenicity in RA, supported by evidence of enhanced complement activation from synovial fluid of RA patients and the ability of ACPA to activate complement via both the classical and alternative pathways (Trouw et al., 2009). Pathogenic autoantibodies have also been linked to RA-associated bone loss through IL-8 mediated

enhancement of osteoclast differentiation (Krishnamurthy et al., 2016).

RA is associated with defective central and peripheral B cell tolerance, contributing to an excess of autoreactive B cells in the mature naive B cell subpool, increased proportion of polyreactive antibodies recognising immunoglobulins and cyclic citrullinated peptides (Samuels et al., 2005b). Notably despite immunosuppressive therapy in RA, post-treatment frequency of autoreactive mature naive B cell clones remains elevated consistent with primary defective early B cell tolerance and a limited ability of current therapeutics to target this (Menard et al., 2011).

Serum levels of BAFF are high in early RA and correlate with titres of IgM rheumatoid factor and anti- cyclic citrullinated peptide autoantibody, as well as with joint involvement; furthermore, levels of BAFF improve in parallel with clinical severity and autoantibody levels in response to methotrexate therapy (Bosello et al., 2008). Notably a cytokine environment conducive to B cell activation and survival has been discerned in very early RA, specifically elevation in BAFF and APRIL (a proliferation- inducing ligand, involved in class-switch recombination and plasma cell differentiation and survival) levels including enrichment in synovial fluid, suggesting a primary role in disease (Moura et al.,

2011). Pathologically, RA articular synovium demonstrates infiltration of plasma cells, positively correlating with synovial fluid levels of APRIL (Dong et al., 2009).

Supporting a key role for T-B interactions in activating autoreactive B cells, T cell promotion of extra- follicular B cell responses as an alternative means of B cell activation via Toll-like receptors amplifies autoantibody production through CD40L and IL-21 signalling (Sweet et al., 2011). Moreover, mice deficient in CXCR5 on T cells are resistant to development of CIA, exhibiting impaired germinal centre formation and failing to mount an IgGl antibody response to CM (Moschovakis et al., 2017). Patients with RA show an expansion in peripheral circulating T _F H cells, correlating with autoantibody titres; notably circulating plasmablast levels in RA correlate with clinical disease activity and markers of inflammation (CRP, ESR) (Nakayamada et al., 2018). In this context plasmablasts may function to present antigen to T cells and promote T cell differentiation, in addition to antibody secretion, thus perpetuating joint inflammation (Nakayamada et al., 2018). Notably, T _F H cells have also been identified within RA synovium as part of the immune infiltrate (Chu et al., 2014), together with regulatory T cells (Tregs) (Penatti et al., 2017). Highlighting a potential pathogenic consequence of the latter, Tregs appear functionally compromised in RA, an effect improved following anti-TNF-a therapy (Ehrenstein et al., 2004). Importantly, while CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Tregs are enriched in inflamed RA synovium, they appear less functional indicating a poorer ability to mediate immune tolerance (Sun et al., 2017). A potential mechanism underlying this observation is that of B cell- derived IFN-g mediated suppression of Treg differentiation, shown to promote autoimmune experimental arthritis in mice (Olalekan et al., 2015).

B cell depletion in RA using rituximab significantly improves symptoms in RA (Edwards et al., 2004), including in patients refractory to anti-TNF-a therapy (Cohen et al., 2006). Rituximab in RA is more effective in seropositive cases (i.e. patients exhibiting ACPA and RF); moreover, positive clinical responses correlate with significant reductions in autoantibodies in parallel with inflammatory markers (Cambridge et al., 2003), as well as the extent of B cell depletion (Vancsa et al., 2013). Autoantibody depletion using immunoadsorption has also proven efficacious in refractory RA (Furst et al., 2000), likely in part to relate to removal of immune complexes and potentially due to removal of complement components (Kienbaum et al., 2009).

Sjogren's syndrome (SjS; Sjorgen's disease)

SjS is a systemic autoimmune disorder which primarily results in inflammation and destruction of exocrine glands by inflammatory infiltrates and IgG plasma cells (especially salivary and lacrimal) with ensuing tissue destruction , but can lead to systemic disease characterised by peri-epithelial infiltration by lymphocytes and immune complex deposition (Brito-Zeron et al 2016). The latter contain T cells, B cells and plasma cells (Hansen et al., 2007). Systemic involvement, e.g. renal disease, is also characterised by marked enrichment of these cells, especially plasma cells (Jasiek et al., 2017).

SjS syndrome is associated with a number of autoantibodies against autoantigens including Ra, La, Fc fragment of IgG and muscarinic M3 receptors. IgG autoantibodies targeting M3 from patients with SjS have been shown to exert an anti-secretory effect in both mouse and human acinar cells, an impact expected to damage salivary production and contribute to the xerostomia (dry mouth) observed in patients (Dawson et al., 2006).

Ectopic formation of germinal centres is recognised in salivary glands in SjS, with B cell-T cell interactions within the germinal centre important to disease pathogenesis and B cell dysregulation (Pontarini et al., 2018). Other evidence for B cell hyperactivity in SjS includes autoantibody production, hypergammaglobulinaemia and increased risk for developing B cell non-Hodgkin's lymphoma (Hansen et al., 2007).

Inflamed salivary glands from patients with SjS show a very significant upregulation in BAFF expression, produced in part from T cells (Lavie et al., 2004), which is also found to be elevated in serum, and expected to promote an environment conducive to autoreactive B cell survival.

Supporting the importance of this regulator of B cell survival and differentiation in SjS, transgenic mice overexpressing BAFF develop sever sialadenitis and submaxillary gland destruction in a phenotype similar to that of human SjS (Groom et al., 2002).

Peripheral circulating T _F H cells are expanded in patients with SjS and also appear in the saliva, the latter correlating with memory B cells and plasma cells suggesting that T _F H cells contribute to the pathophysiology of SjS by promoting B cell maturation (Jin et al., 2014). Notably an increase in salivary plasma cell content is positively correlated with serum ANA levels in SjS (Jin et al., 2014). Illustrating the importance of B cell-T cell crosstalk mechanistically in SjS, B cell depletion using rituximab lowers circulating T _F H cell levels, IL-17 producing CD4 ⁺ T cells and serum IL-21 and IL-17, with reductions in circulating T _F H cells associating with lower clinical measures of disease activity (Verstappen et al., 2017).

B cell depletion using rituximab has some evidence of effect clinically in SjS, including improvement in salivary gland ultrasound score (Fisher et al., 2018). Supporting a role for enhanced B cell activation in SjS, targeting BAFF using belimumab has efficacy in reducing an index of clinical activity (Mariette et al., 2015). Graft-versus-host disease (GVHD)

GVHD is the most frequent life-threatening complication of allogeneic haematopoietic stem cell transplantation. While the immunopathogenesis and initiation of acute GVHD is thought to be driven by immunocompetent T cells in the donated graft tissue recognising the new host as foreign leading to immune activation and attack (Zeiser and Blazar, 2017), there is a significant role for B cells particularly in chronic GVHD.

Underlining defects in B cell homeostasis in GVHD, B cell derived antibodies against

histocompatibility antigens (also targets of donor T cells) are evident in GVHD and correlated with disease (Miklos et al., 2005). In both acute and chronic forms of GVHD, dermo-epidermal immunoglobulin deposits in association with C3 complement deposition are observed (Tsoi et al., 1978). Murine models of GVHD have also demonstrated an ability of antibodies from donor B cells to damage the thymus and peripheral lymphoid organs in association with cutaneous pathogenic TH17 infiltration to augment GVHD (Jin et al., 2016).

Patients with chronic GVHD display significantly increased BAFF/B cell ratios compared to patients without GVHD and healthy donors (Sarantopoulos et al., 2009). Notably increased BAFF levels in serum correlate with increases in both circulating pre-germinal centre B cells and plasmablasts (Sarantopoulos et al., 2009). Notably, B cells from patients with chronic GVHD exhibit a heightened metabolic state together with reduced pro-apoptotic signalling priming them for survival (Allen et al., 2012).

Studies in a murine model of chronic GVHD and bronchiolitis obliterans reveal robust germinal centre reactions at the time of disease initiation, organ fibrosis associated with infiltration of B220+

B cells and CD4+ T cells together with alloantibody deposition (Srinivasan et al., 2012).

Substantiating the key role of germinal centre formation, the associated follicular T-B cell interaction and pathogenic alloantibody formation, blockade of germinal centre formation suppresses the development of GVHD (Srinivasan et al., 2012). Similarly, depletion of donor splenocyte CD4 ⁺ T cells in a mouse model of GVHD prevents aberrant germinal centre formation and T _FH and germinal centre B cells, while allogeneic splenocytes depleted of B220 ⁺ B cells also reduced excessive development of both germinal centre B cells and T _FH cells, underlining their interdependence (Shao et al., 2015).

B cell depletion using rituximab has proven effective as first line treatment of chronic GVHD, in association with a reduction in circulating ICOS ^hl PD-l ^hl T _F H cells (Malard et al., 2017). Eosinophilic oesophagitis (EO)

EO is a chronic allergen-driven immune mediated disorder characterised pathologically by prominent eosinophilic infiltration (Chen and Kao, 2017). An important role for T helper type 2 cells (Th2) has been identified in response to allergens and associated production of IL-4, IL-5 and IL-13, with pathogenesis thought to be driven by a combination of IgE-mediated and non-lgE-mediated mechanisms (Weinbrand-Goichberg et al 2013). Eosinophils promote inflammation, activate smooth muscle and induce mast and basophil cell degranulation (Chen and Kao, 2017).

Oesophageal biopsies from patients with EO reveal increased density of B cells and IgE-bound mast cells versus controls, with a positive correlation between CD20+ B cell density and mast cells (Vicario et al., 2010). Notably, an upregulation in expression of IgE heavy chain and mature IgE mRNA has been identified with evidence of local class-switch recombination (CSR) to IgE provided by detection of germline transcripts for e, m and y4, in addition to expression of AID catalysing the initial step of CSR (Vicario et al., 2010). These findings indicate active B cell recruitment in EO, together with local CSR and mature IgE production in both 'atopic' and 'non atopic' individuals, indicating a clear local antibody response in EO (Vicario et al., 2010). Other studies have also identified increases in IgG subclasses, IgA and IgM in EO, particularly lgG4, the latter correlating with oesophageal eosinophil counts, histology and stage of disease (Rosenberg et al., 2018). Immune complex formation and lgG4 ⁺ plasma cells have been noted in the deep lamina propria (Clayton et al., 2014). Importantly, lgG4-switched B cells can switch to IgE but the reverse does not occur due to genetic deletion during the process of CSR to IgE. Notably the transition to a plasma cell phenotype occurs early with IgE B cells and significantly more so than with IgG-switched B cells (Aalberse et al., 2016).

A role for IgE is further suggested by the observation of IgE-bearing cells including mast cells in EO (Straumann et al., 2001). Notably, cells expressing the high affinity receptor for IgE, FCERI, are present in large numbers in the oesophageal epithelium of patients with EO, suggesting this receptor to be important in IgE-mediated activation of immune cells in EO (Yen et al., 2010).

EO is often associated with IgE sensitisation to allergens in food in children and plant/aero allergens in adults. Serum IgE levels are also often significantly elevated in patients with EO, consistent with the presence of IgE-producing long-lived plasma cells (Aalberse et al., 2016), which together with specific IgE antibodies suggest a contribution of IgE to the pathogenesis (Straumann et al., 2001). Notably food-specific IgE antibodies predict oesophageal eosinophilia in children (Erwin et al., 2017). Active oesophagitis in EO is associated with elevated oesophageal levels of plasma cells (Mohammad et al., 2018). Notably, in addition to immunoglobulin targeting exogenous antigens, recent data suggest the presence of autoantibodies in EO, specifically anti-NC16A which appear to correlate with histological response (Dellon et al., 2018).

Supporting a pathogenic role for IgE in EO, a pilot study of anti-lgE treatment using omalizumab demonstrated clinical improvement in a proportion of patients, accompanied by lower tissue IgE levels, tryptase positive cells and eosinophils (Loizou et al., 2015).

Atopic asthma (extrinsic, early-onset or allergic asthma)

Atopic asthma I associated with atopy (allergic rhinitis and atopic eczema) and thought to be driven substantially by a type 2 helper T cell (TH2) response to promote an early IgE-mediated (i.e. type 1) hypersensitivity reaction, together with IL-5 promoted activation of eosinophils.

Notably TH2 cells secrete high levels of IL-4 and IL-13 which promote IgE class-switching by B cells. Subsequently, IgE memory B cells can differentiate into plasma cells to produce specific IgE that can bind to its high affinity receptor, FceRI, on target cells such as mast cells and basophils (Palomares et al., 2017). Binding of antigen-specific IgE to FceRI on mast cells is critical in sensitising these cells to release mast cell mediators in a specific manner. In addition, immune complexes formed from antigen-lgE can bind to CD23 on B cells or FceRI to further amplify IgE-associated immune responses (Galli and Tsai, 2012). Importantly, bronchial epithelial cells from some patients with asthma, but not healthy controls, express FceRI, are capable of fixing IgE and functional in terms of eicosanoid release (Campbell et al., 1998).

Importantly, class switch recombination from IgM/lgG/lgA to IgE can occur locally in bronchial tissue in asthma, resulting in clonal selection and affinity maturation of IgE-producing B cells to release IgE locally (Takhar et al., 2007). Patients with allergic asthma exhibit highly elevated levels of lgE ⁺ CD19 ⁺ B cells in the airways compared to healthy controls and 'allergic' controls, as well as increased lgE ⁺ memory B cells and lgE ⁺ plasma cells (Oliveria et al., 2017). Notably, the frequency of lgE+ B cells corelates positively with airway levels of eosinophils, IgE and BAFF, findings consistent with local maturation and proliferation of lgE ⁺ B cells in the airways of patients with allergic asthma (Oliveria et al., 2017) to drive the disease process through their production of IgE and potent antigen- presentation function (Wypych et al., 2018). Furthermore, tissue resident memory B cells have also been identified in the airways of a mouse model of allergic asthma, providing a resident B cell population that can be rapidly locally activated in response to allergen/antigen re-exposure (Turner et al., 2017). Children with asthma and/or atopy show evidence of expanded lgE ⁺ plasmablasts in addition to lgE ⁺ memory cells and TH2 cells; notably plasmablast numbers positively correlate with frequency of circulating TH2 cells (Heeringa et al., 2018).

Substantiating a key role for B cells in the pathogenesis of atopic asthma, B cell depletion using anti- CD20 before house dust mite (HDM) challenge in HDM sensitised mice markedly reduces the allergic response, with reduced CD4 ⁺ CD44 ⁺ T cells, eosinophils and neutrophils in lung immune infiltrates consistent with a lower T _H 2 response (Wypych et al., 2018). B cells thus play a critical role in amplifying TH2 responses in vivo promote the allergic response, this is likely to in part reflect their ability to efficiently present antigen (Wypych et al., 2018).

Substantiating a role for IgE in atopic/allergic asthma, administration of monoclonal antibody therapy targeting FceRI (thereby inhibiting the binding of endogenous IgE to mast and other effector cells without stimulating mast cell activation) to allergic asthmatic patients suppresses early and late phase responses to inhaled allergen, associated with lowering of serum IgE, blunting of sensitivity to inhaled allergen and attenuation of the fall in respiratory capacity associated with allergen inhalation (Fahy et al., 1997). Further evidence for the critical pathogenic role of IgE in persistent asthma comes from a trial of omalizumab, a humanised monoclonal anti-lgE antibody, in inner city children and young adults. This demonstrated a significant reduction in burden of asthma symptoms, frequency of exacerbations, particularly seasonal peaks, despite a lower requirement for inhaled glucocorticoid and b-agonist bronchodilator therapy (Busse et al., 2011). Specific extracorporeal immunoadsorption of IgE has also demonstrated efficacy in reducing allergen-specific basophil sensitivity and clinical symptoms during pollen season in patients with allergic asthma (Lupinek et al., 2017). Unlike omalizumab administration, this approach while invasive is not limited by threshold levels of IgE (Lupinek et al., 2017).

Atopic dermatitis (AD; atopic eczema)

AD is a chronic inflammatory skin disorder characterised by pruritic eczematous skin lesions. It is associated with other stopic diseases (asthma and allergic rhinitis), with shared aspects of pathophysiology, in particular a propensity to form IgE antibodies and sensitisation to exogenous triggers (Zheng et al., 2011). AD is considered a biphasic T cell-mediated disorder with a TH2 to THI switch promoting chronicity, in addition to a significant disease component driven by B cell derived IgE (Furue et al., 2017). Notably ~80-90% of patients with AD feature raised serum IgE, with elevated levels correlating with IgE autoreactivity against multiple antigens (Furue et al., 2017). The enhanced levels of IgE are thought to primarily reflect greater numbers of IgE antibody-producing cells (Thomas et al., 1995).

Patients with AD display IgE autoantibodies against keratinocyte proteins, particularly in severe cases (Altrichter et al., 2008). The correlation of autoreactive IgE in AD with clinical severity and absence in other skin disorders supports a role for IgE-mediated autoreactivity in disease pathogenesis (Navarrete-Dechent et al., 2016). Notably AD also occurs in association with other autoimmune diseases, e.g. vitiligo (Mohan and Silverberg, 2015), with a proportion of patients with severe facial rashes exhibiting ANA positivity (Higashi et al., 2009), suggest more generalised humoral immune dysregulation in AD. Autoantibodies described include those targeting SART-1, cytokeratin type II, hMnSOD and BCL7B amongst others (Navarrete-Dechent et al., 2016). Clinical severity scores correlate strongly with some of these specific IgE autoantibodies (Schmid- Grendelmeier et al., 2005). The allergenicity of this antigen is further supported by its ability to induce T-cell proliferation and positive immediate responses to skin challenge (Schmid-Grendelmeier et al., 2005).

Further supporting a role for B cells in AD, analysis of peripheral lymphocyte subsets has revealed B cell alterations particularly in children with AD, with positive correlations between activated B cells and memory T cell levels (Czarnowicki et al., 2017). Furthermore, peripheral blood analysis indicates that early AD is characterised by aberrant B cell maturation and reveals a positive correlation between memory B cells and THI and TH2 cells in AD (Czarnowicki et al., 2017). Importantly, in children with AD IgE sensitisation is seen to cluster with total IgE levels, switched memory B cells and TH1/TH2 cells, with evidence of accelerated B cell development that would support IgE class switching (Czarnowicki et al., 2017).

In adults, AD is associated with an increase in circulating transitional B cells, chronically activated memory B cells, plasmablasts and IgE memory B cells (Czarnowicki et al., 2016). Notably circulating cell expression of CD23, the low-affinity receptor for the Fc region of IgE (FceRII), is increased in AD and correlates with AD clinical severity (Czarnowicki et al., 2016); this observation is notable given the role of CD23 to promote IgE synthesis/responses (Pene, 1989).

Supporting a major role for B cells in the pathogenesis of AD, B cell depletion using rituximab results in substantial clinical improvement (severity/area affected), in conjunction with improvements in histology (reduced B and T cell infiltration), IL-5/IL-13 and some reduction in total IgE (Simon et al., 2008). Notably, despite near complete depletion of circulating B cells, those in the skin were less substantially reduced (by ~50%), with plasma cells also evident in skin samples both before and after therapy (Simon et al., 2008). Furthermore, sequential treatment of severe refractory AD using anti- IgE (omalizumab) followed by B cell depletion (rituximab) has reported dramatic clinical responses in conjunction with lowering of serum IgE and peripheral blood B cell levels (Sanchez-Ramon et al., 2013). Further supporting a role for pathogenic IgE immunoglobulin in driving AD, repeated IgE immunoadsorption in patients with AD and increased serum IgE results in significant clinical improvement together with lowering of IgE (Daeschlein et al., 2015).

Churg-Strauss syndrome (Churg-Strauss vasculitis; eosinophilic granulomatosis with polyangiitis; CSS/EG PA)

Churg-Strauss syndrome, also known as eosinophilic granulomatosis with polyangiitis (EGPA), is a small-medium vessel systemic necrotising vasculitis, part of the clinical spectrum of ANCA-associated vasculitis (ANCA, antineutrophil cytoplasm antibody positive in ~40) and associated with severe adult-onset asthma, sinusitis and blood/tissue eosinophilia (Groh et al., 2015).

The pathogenesis of CSS involves T cells (particularly excessive TH2 responses but also involvement of THI and reduced regulatory T cells), activated tissue eosinophils and B cells with a humoral response (Greco et al., 2015). ANCAs are directly pathogenic and primarily target myeloperoxidase (MPO) and proteinase 3, with the former characteristic for CCS. ANCA result in neutrophil activation and degranulation leading to cytokine, cytolytic enzyme and ROS release through binding of ANCA- specific antigens and, via their Fc region, the Fey receptor on neutrophils (Nakazawa et al., 2019).

Supporting a role for IgE in pathogenesis of CSS, mice subjected to a cutaneous reverse passive Arthus reaction (using IgE) to provide an IgE-immune complex challenge develop cutaneous eosinophilic vasculitis reminiscent of CSS (Ishii et al., 2009). Notably eosinophil infiltration in this model is strikingly specific for IgE-mediated immune complex challenge and barely seen with IgG antibody injection (Ishii et al., 2009). Evidence also exists to support pathogenicity of IgE in CSS via immune complex formation and activation of complement (Manger et al., 1985).

Patients with CSS who exhibit active disease and frequent relapse show increased levels of activated B cells and reduced levels of circulating T regulatory cells (Tsurikisawa et al., 2013). Patients with CSS also demonstrate a cellular milieu conducive to plasma cell differentiation and antibody-mediated responses through an increase in IL-21 secreting T helper cells, specifically in ANCA positive patients (Abdulahad et al., 2013).

Supporting a key role for B cells and their autoantibodies in the pathogenesis of CCS, B cell depletion using rituximab is clinically effective in inducing remission or partial responses and lowering of requirement for corticosteroid therapy; notably levels of baseline ANCA associate with higher levels of remission (Mohammad et al 2016). These findings have been confirmed in another clinical study of refractory patients with CSS, with rituximab lowering levels of IgE, CRP and eosinophils in conjunction with inducing remission (Thiel et al., 2017). There is also evidence for a corticosteroid sparing effect of targeting IgE using omalizumab in refractory/relapsing CCS (Jachiet et al., 2016).

Allergic rhinitis (AR)

AR is a common and chronic IgE-mediated inflammatory nasal disorder frequently associated with other atopic features (asthma and atopic dermatitis). Exposure to specific allergens promotes allergen-specific IgE production which can then bind to target cells (e.g. mast cells and basophils) via the high affinity receptor, FceRI (Wise et al., 2018). In turn, nasal mast cells from patients with AR exhibit upregulation of FceRI expression and increased cell-bound IgE correlating with serum IgE levels; these cells can also induce IgE production by B cells indicating a feed-forward IgE- FceRI - mast cell axis critically dependent on pathogenic IgE that can perpetuate AR (Pawankar and Ra, 1998).

Nasal mucosal B cells are over 1000-fold more frequent than in peripheral blood in AR and produce IgE following allergen exposure (Coker et al., 2003; Takhar et al., 2005) . There is evidence supporting local class switch recombination in nasal mucosa of patients with AR (Cameron et al., 2000), suggesting that tissue resident/local B cells under Ig isotype switching to IgE in the context of local immune responses to allergen (Cameron et al., 2003). Both lgE+ B cells and lgE+ plasma cells are enriched in the nasal mucosa of patients with AR (KleinJan et al., 2000).

Substantiating a central role for IgE in AR, anti-lgE therapy using omalizumab is clinically effective in patients with AR, also inhibiting seasonal associated allergen-induced increases in tissue/blood eosinophils (Flolgate et al., 2005; Tsabouri et al., 2014).

Allergic eye disease

Seasonal and perennial allergic conjunctivitis are the commonest forms of allergic eye disease and are associated with other atopic diseases with mechanisms similar to those outlined, including an important role for B cell derived IgE. Supporting this, anti-lgE therapy using omalizumab has shown efficacy in atopic individuals with coexisting eye disease (Kopp et al., 2009).

Chronic non-autoimmune urticaria (chronic spontaneous urticaria, CSU)

Urticaria is a common, mast cell-driven disease, and can be classified as acute or chronic; chronic non-autoimmune urticaria can itself be classified as chronic spontaneous urticaria (CSU) and chronic inducible urticaria (Radonjic-Floesli et al., 2018). Although there are no obvious external triggers in CSU, and most patients have an autoimmune cause, there is a significant proportion of patients that do not have an autoimmune disease. In these, IgE binding to FceRI on mast cells without cross- linking is thought to promote survival and proliferation of mast cells, decrease the threshold for mast cell mediator release (Chang et al., 2015). Consistent with this and a pathogenic role for B cell- derived IgE in this condition, to date, there have been 2 phase II and 4 phase III randomized, placebo-controlled clinical trials that have convincingly established that IgE depletion using anti-lgE therapy with omalizumab is efficacious and safe for treating CSU that is refractory to the current standard care (Chang et al., 2015).

Thus, in an embodiment, the invention provides (i) a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease in a subject and (ii) a method of treatment or prevention of a pathogenic immunoglobulin driven B cell disease in a subject by administering to said subject an effective amount of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof wherein in the case of (i) and (ii) the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, autoimmune alopecia, vitiligo, dermatitis herpetiformis, chronic autoimmune urticaria, coeliac disease, Graves' disease, Hashimoto's thyroiditis, Type 1 diabetes mellitus, autoimmune Addison's disease, autoimmune haemolytic anaemia, autoimmune thrombocytopenic purpura,

cryoglobulinemia, pernicious anaemia, myasthenia gravis, multiple sclerosis, neuromyelitis optica, autoimmune epilepsy and encephalitis, autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis.

Preferably the pathogenic IgG driven B cell disease is selected from pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, immune thrombocytopenia (ITP/AITP), myasthenia gravis or variants thereof.

Exemplary pathogenic IgA driven B cell diseases may be selected from the group consisting of the skin related diseases dermatitis herpetiformis, linear IgA disease, pemphigus vulgaris, pemphigus foliaceus, cicatricial pemphigoid and bullous pemphigoid. Alternatively, the disease may be the gut related disease coeliac disease. Alternatively, the disease may be the kidney related disease IgA nephropathy.

Preferably the pathogenic IgA driven B cell disease is selected from dermatitis herpetiformis, linear IgA disease and IgA nephropathy. Clozapine is associated with high levels of CNS penetration which could prove to be a valuable property in treating some of these diseases (Michel et al., 2015).

In certain diseases, more than one Ig type (such as IgG and IgA) may play a role in the pathology of the disease. For example, in dermatitis herpetiformis, coeliac disease, pemphigus vulgaris, pemphigus foliaceus, cicatricial pemphigoid and bullous pemphigoid, production of pathogenic IgA is thought to contribute towards the pathology as well as IgG.

In certain diseases, such as multiple sclerosis, vitiligo, Type 1 diabetes mellitus, autoimmune Addison's disease, dermatitis herpetiformis, coeliac disease, primary biliary cirrhosis, primary sclerosing cholangitis and autoimmune thrombocytopenic purpura there may also be a T cell component that contributes towards the pathology of the disease. This arises because B cells act as professional antigen-presenting cells for T cells (their importance is increased also due to their sheer numbers). B cells secrete significant amounts of cytokines that impact T cells. B-T interaction is involved in responses to T dependent protein antigens and class switching. Therefore, clozapine and norclozapine or pegylated derivatives thereof are expected to have an effect on T cells in part due to their effect on reducing B cell numbers.

The invention also provides (i) a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component in a subject and (ii) a method of treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component in a subject by administering to said subject an effective amount of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof wherein in the case of (i) and (ii) the pathogenic immunoglobulin driven B cell disease with a T cell component is a disease selected from the group consisting of vitiligo, psoriasis, coeliac disease, dermatitis herpetiformis, discoid lupus erythematosus, dermatomyositis, polymyositis, Type 1 diabetes mellitus, autoimmune Addison's disease, multiple sclerosis, interstitial lung disease, Crohn's disease, ulcerative colitis, thyroid autoimmune disease, autoimmune uveitis, primary biliary cirrhosis, primary sclerosing cholangitis, undifferentiated connective tissue disease, autoimmune thrombocytopenic purpura, mixed connective tissue disease, an immune-mediated inflammatory disease (IMID) such as scleroderma, rheumatoid arthritis, Sjogren's disease, an autoimmune connective tissue disease such as systemic lupus erythematosus and graft versus host disease.

In certain diseases, specific Ig types (such as IgG, IgA) are believed to play a role in the pathology of the disease. For example, in dermatitis herpetiformis and coeliac disease, production of pathogenic IgG and IgA are thought to contribute towards the pathology. For example, in multiple sclerosis, vitiligo, autoimmune Addison's disease, type I diabetes mellitus, primary biliary cirrhosis, primary sclerosing cholangitis pathogenic and autoimmune thrombocytopenic purpura, IgG is thought to contribute towards the pathology. The finding by the inventors that clozapine significantly reduces class switched memory B cells and will consequently reduce the numbers of ASCs and the secretion of specific immunoglobulins means that pathogenic IgG levels and pathogenic IgA levels should be reduced. The present inventors have also discovered that clozapine reduces total IgG levels and total IgA levels.

In one embodiment the pathogenic immunoglobulin is pathogenic IgG. In one embodiment the pathogenic immunoglobulin is pathogenic IgA. In one embodiment the pathogenic immunoglobulin is pathogenic IgM.

Preferably, the pathogenic immunoglobulin driven B cell disease with a T cell component is psoriasis, an autoimmune connective tissue disease such as systemic lupus erythematosus, an immune- mediated inflammatory disease (IMID) such as scleroderma, rheumatoid arthritis or Sjogren's disease, or neurological autoimmune diseases such as multiple sclerosis, NMO or autoimmune epilepsy.

The invention further provides for (i) a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic IgE driven B cell disease in a subject and (ii) a method of treatment or prevention of a pathogenic IgE driven B cell disease in a subject by administering to said subject an effective amount of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof wherein in the case of (i) and (ii) the pathogenic IgE driven B cell disease is a disease selected from the group consisting of atopic asthma, atopic dermatitis, chronic non- autoimmune urticaria, Churg-Strauss vasculitis, allergic rhinitis and allergic eye disease preferably atopic dermatitis, atopic asthma, allergic rhinitis and eosinophilic esophagitis.

Preferably the disease is selected from atopic dermatitis, atopic asthma, allergic rhinitis and Churg- Strauss vasculitis.

In certain diseases, such as atopic dermatitis, there may also be a T cell component that contributes towards the pathology of the disease. This arises because B cells act as professional antigen- presenting cells for T cells (their importance is increased also due to their sheer numbers). B cells secrete significant amounts of cytokines that impact T cells. B-T interaction is involved in responses to T dependent protein antigens and class switching. Therefore, clozapine and norclozapine are expected to have an effect on T cells due to their effect on reducing B cell numbers.

Suitably the compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof inhibits mature B cells, especially CSMBs and plasmablasts, particularly CSMBs. "Inhibit" means reduce the number and/or activity and/or responsivity of said cells. Thus, suitably clozapine or norclozapine or pegylated derivatives thereof reduces the number of CSMBs and plasmablasts, particularly CSMBs.

In an embodiment the compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof has the effect of decreasing CD19 (+) B cells and/or CD19 (-) B-plasma cells.

The term "treatment" means the alleviation of disease or symptoms of disease. The term

"prevention" means the prevention of disease or symptoms of disease. Treatment includes treatment alone or in conjunction with other therapies. Treatment embraces treatment leading to improvement of the disease or its symptoms or slowing of the rate of progression of the disease or its symptoms. Treatment includes prevention of relapse.

The term "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. Example dosages are discussed below.

As used herein, a "subject" is any mammal, including but not limited to humans, non-human primates, farm animals such as cattle, sheep, pigs, goats and horses; domestic animals such as cats, dogs, rabbits; laboratory animals such as mice, rats and guinea pigs that exhibit at least one symptom associated with a disease, have been diagnosed with a disease, or are at risk for developing a disease. The term does not denote a particular age or sex. Suitably the subject is a human subject.

The compound used according to the invention may be pegylated clozapine, pegylated norclozapine or a pharmaceutically acceptable salt or solvate thereof. It will be appreciated that for use in medicine the salts of clozapine and norclozapine and pegylated derivatives thereof should be pharmaceutically acceptable. Suitable pharmaceutically acceptable salts will be apparent to those skilled in the art. Pharmaceutically acceptable salts include those described by Berge, Bighley and Monkhouse J. Pharm. Sci. (1977) 66, pp 1-19. Such

pharmaceutically acceptable salts include acid addition salts formed with inorganic acids e.g.

hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or

naphthalenesulfonic acid. Other salts e.g. oxalates or formates, may be used, for example in the isolation of clozapine and are included within the scope of this invention.

A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof may be prepared in crystalline or non-crystalline form and, if crystalline, may optionally be solvated, e.g. as the hydrate. This invention includes within its scope stoichiometric solvates (e.g. hydrates) as well as compounds containing variable amounts of solvent (e.g. water).

A "prodrug", such as an N-acylated derivative (amide) (e.g. an N-acylated derivative of norclozapine) is a compound which upon administration to the recipient is capable of providing (directly or indirectly) clozapine or an active metabolite or residue thereof. Other such examples of suitable prodrugs include alkylated derivatives of norclozapine other than clozapine itself.

The compound used according to the invention may be a pegylated derivative of clozapine or a pegylated derivative of norclozapine. The compound used according to the invention may be a prodrug of a pegylated derivative of clozapine or a prodrug of a pegylated derivative of norclozapine. The compound used according to the invention may be a pharmaceutically acceptable salt or solvate (or salt of solvate) of any of the aforesaid. Pegylated derivatives of clozapine and norclozapine are derivatives of clozapine and norclozapine which bear as a substituent a PEG group. A PEG group is a polyethylene glycol group which may have a capped or terminal OH group and may be straight chain or branched but is preferably straight chain. Suitable capping groups including alkyl groups such as Cl-4alkyl groups e.g. methyl, hydroxalkyl groups such as Cl-4hydroxyalkyl and acyl groups such as C(=0)Cl-15alkyl e.g. C(=0)Cl-3alkyl. Unbranched polyethylene glycols typically have formula -(O)y- (CH2-CH2-0)n-H where n can be 2-50 e.g. a-b and y is 1 when the PEG groups is attached to a carbon atom and y is 0 when the PEG groups is attached to nitrogen atom. The terminal H may be replaced by a capping group. The point of attachment of the substituent PEG group to norclozapine may, for example be the free piperazine nitrogen atom. A number of exemplary pegylated derivatives of clozapine and norclozapine are disclosed in GB1562874 (Abbott) herein incorporated by reference in its entirety.

Thus, exemplary pegylated derivatives of clozapine and norclozapine that may be used according to the invention and are disclosed in GB1562874 have the following formula (A):

wherein:

R represents a hydrogen atom, a lower alkyl group, a hydroxylower alkyl group or -C(0)Ri; and

Ri represents an alkyl group having 1 to 15 carbon atoms.

Specific individual compounds that may be mentioned are compounds of formula (A) in which: (a) R=H (see Example 1 of GB1562874);

(b) R=methyl (see Example 2 of GB1562874);

(c) R=CH2CH20H (see Example 3 of GB1562874);

(d) R = C(=0)(CH2)i ₅ (see Example 4 of GB1562874);

Pegylated derivatives of clozapine and norclozapine (and prodrugs thereof) may, for example, be prepared by methods disclosed in GB1562874 or by other methods known to a skilled person.

Use of a pegylated derivative of clozapine, norclozapine or a prodrug thereof may have the advantage of improved physicochemical (e.g. solubility to increase cellular drug availability) and physiological characteristics compared to the parent molecule, improved pharmacokinetic profile to increase circulating half-life/exposure while retaining efficacy (potentially allowing infrequent or less frequent dosing or more sustained clinical response), increasing oral bioavailability, favourable modification of biodistribution including impeding blood brain penetration/reduced central nervous system exposure or exclusion thereby minimising centrally-driven adverse effects and potentially enabling higher dosing with enhanced therapeutic index, overall improved pharmacodynamic profile resulting in increased efficacy, an improved safety profile with reduced toxicity and better patient compliance.

Isotopically-labelled compounds which are identical to clozapine or norclozapine but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature, or in which the proportion of an atom having an atomic mass or mass number found less commonly in nature has been increased (the latter concept being referred to as "isotopic enrichment") are also contemplated for the uses and method of the invention. Examples of isotopes that can be incorporated into clozapine or norclozapine include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, iodine and chlorine such as ² H (deuterium), ³ H, C, ¹³ C, ¹⁴ C, ¹⁸ F, ¹²³ l or ¹²⁵ l, which may be naturally occurring or non- naturally occurring isotopes.

Clozapine or norclozapine or pegylated derivatives thereof and pharmaceutically acceptable salts of clozapine or norclozapine or pegylated derivatives thereof that contain the aforementioned isotopes and/or other isotopes of other atoms are contemplated for use for the uses and method of the present invention. Isotopically labelled clozapine or norclozapine or pegylated derivatives thereof, for example clozapine or norclozapine into which radioactive isotopes such as ³ H or ¹⁴ C have been incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e. ³ H, and carbon-14, i.e. ¹⁴ C, isotopes are particularly preferred for their ease of preparation and detectability. C and ¹⁸ F isotopes are particularly useful in PET (positron emission tomography).

Since clozapine or norclozapine or pegylated derivatives thereof are intended for use in

pharmaceutical compositions it will readily be understood that it is preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions.

In general, clozapine or norclozapine or pegylated derivatives thereof may be made according to the organic synthesis techniques known to those skilled in this field (as described in, for example, US3539573).

A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in therapy is usually administered as a pharmaceutical composition. Also provided is a pharmaceutical composition comprising clozapine or norclozapine, or a pharmaceutically acceptable salt and/or solvate and/or prodrug thereof and a pharmaceutically acceptable diluent or carrier. Said composition is provided for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease, a pathogenic immunoglobulin driven B cell disease with a T cell component or a pathogenic IgE driven B cell disease in a subject wherein said compound causes mature B cells to be inhibited in said subject.

A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof may be administered by any convenient method, e.g. by oral, parenteral, buccal, sublingual, nasal, rectal or transdermal administration, and the pharmaceutical compositions adapted accordingly. Other possible routes of administration include intratympanic and intracochlear. Suitably, a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof are administered orally.

A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof which are active when given orally can be formulated as liquids or solids, e.g. as syrups, suspensions, emulsions, tablets, capsules or lozenges.

A liquid formulation will generally consist of a suspension or solution of the active ingredient in a suitable liquid carrier(s) e.g. an aqueous solvent such as water, ethanol or glycerine, or a non- aqueous solvent, such as polyethylene glycol or an oil. The formulation may also contain a suspending agent, preservative, flavouring and/or colouring agent.

A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations, such as magnesium stearate, starch, lactose, sucrose and cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures, e.g. pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), e.g. aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.

Typical parenteral compositions consist of a solution or suspension of the active ingredient in a sterile aqueous carrier or parenterally acceptable oil, e.g. polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then

reconstituted with a suitable solvent just prior to administration.

Compositions for nasal or pulmonary administration may conveniently be formulated as aerosols, sprays, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a pharmaceutically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container which can take the form of a cartridge or refill for use with an atomising device.

Alternatively, the sealed container may be a disposable dispensing device such as a single dose nasal or pulmonary inhaler or an aerosol dispenser fitted with a metering valve. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas e.g. air, or an organic propellant such as a fluorochlorohydrocarbon or hydrofluorocarbon. Aerosol dosage forms can also take the form of pump-atomisers.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles where the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatine and glycerine.

Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

Compositions suitable for topical administration to the skin include ointments, gels and patches.

In one embodiment the composition is in unit dose form such as a tablet, capsule or ampoule.

Compositions may be prepared with an immediate release profile upon administration (i.e. upon ingestion in the case of an oral composition) or with a sustained or delayed release profile upon administration.

For example, a composition intended to provide constant release of clozapine over 24 hours is described in W02006/059194 the contents of which are herein incorporated in their entirety.

The composition may contain from 0.1% to 100% by weight, for example from 10 to 60% by weight, of the active material, depending on the method of administration. The composition may contain from 0% to 99% by weight, for example 40% to 90% by weight, of the carrier, depending on the method of administration. The composition may contain from 0.05mg to lOOOmg, for example from l.Omg to 500mg, of the active material (i.e. clozapine or norclozapine or pegylated derivatives thereof), depending on the method of administration. The composition may contain from 50 mg to 1000 mg, for example from lOOmg to 400mg of the carrier, depending on the method of administration. The dose of clozapine or norclozapine or pegylated derivatives thereof used in the treatment or prevention of the aforementioned diseases will vary in the usual way with the seriousness of the diseases, the weight of the sufferer, and other similar factors. However, as a general guide suitable unit doses of clozapine as free base may be 0.05 to 1000 mg, more suitably 1.0 to 500mg, and such unit doses may be administered more than once a day, for example two or three a day. Such therapy may extend for a number of weeks or months.

A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof may be administered in combination with another therapeutic agent for the treatment of pathogenic immunoglobulin driven B cell diseases (e.g. IgG, IgE or IgA driven B cell disease), such as those that inhibit B cells and/or T cells and/or B cell - T-cell interactions. Other therapeutic agents include for example: anti-TNFa agents (such as anti-TNFa antibodies e.g. infliximab or adalumumab), calcineurin inhibitors (such as tacrolimus or cyclosporine), antiproliferative agents (such as mycophenolate e.g. as mofetil or sodium, or azathioprine), general anti-inflammatories (such as hydroxychloroquine or NSAIDS such as ketoprofen and colchicine), mTOR inhibitors (such as sirolimus), steroids (such as prednisone), anti-CD80/CD86 agents (such as abatacept), anti-CD-20 agents (such as anti-CD-20 antibodies e.g. rituximab). anti- BAFF agents (such as anti- BAFF antibodies e.g. tabalumab or belimumab, or atacicept), immunosuppressants (such as methotrexate or cyclophosphamide), anti-FcRn agents (e.g. anti-FcRn antibodies), anti-lgE antibodies (e.g. omalizaumab) and other antibodies (such as ARGX-113, PRN-1008, SYNT-001, veltuzumab, ocrelizumab, ofatumumab, obinutuzumab, ublituximab, alemtuzumab, milatuzumab, epratuzumab and blinatumomab). Rituximab may be mentioned in particular.

Other therapies that may be used in combination with the invention include non-pharmacological therapies such as intravenous immunoglobulin therapy (IVIg), subcutaneous immunoglobulin therapy (SCIg) e.g. facilitated subcutaneous immunoglobulin therapy, plasmapheresis and immunoadsorption.

Thus the invention provides a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease in combination with a second or further therapeutic agent for the treatment or prevention of a pathogenic immunoglobulin driven B cell disease (e.g. IgG or IgA driven B cell disease), a pathogenic immunoglobulin driven B cell disease with a T cell component or a pathogenic IgE driven B cell disease e.g. a substance selected from the group consisting of anti-TNFa agents (such as anti-TNFa antibodies e.g. infliximab or adalumumab), calcineurin inhibitors (such as tacrolimus or

cyclosporine), antiproliferative agents (such as mycophenolate e.g. as mofetil or sodium, or and azathioprine), general anti-inflammatories (such as hydroxychloroquine and NSAIDS such as ketoprofen and colchicine), mTOR inhibitors (such as sirolimus), steroids (such as prednisone), anti- CD80/CD86 agents (such as abatacept), anti-CD-20 agents (such as anti-CD-20 antibodies e.g.

rituximab). anti- BAFF agents (such as anti- BAFF antibodies e.g. tabalumab or belimumab, or atacicept), immunosuppressants (such as methotrexate or cyclophosphamide), anti-FcRn agents (e.g. anti-FcRn antibodies) and other antibodies (such as ARGX-113, PRN-1008, SYNT-001, veltuzumab, ocrelizumab, ofatumumab, obinutuzumab, ublituximab, alemtuzumab, milatuzumab, epratuzumab and blinatumomab). Rituximab may be mentioned in particular.

When a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof is used in combination with other therapeutic agents, the compounds may be administered separately, sequentially or simultaneously by any convenient route.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. The individual components of combinations may also be administered separately, through the same or different routes. For example, a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof and the other therapeutic agent may both be administered orally. Alternatively, a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof may be administered orally and the other therapeutic agent via may be administered intravenously or subcutaneously.

Typically, a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof is administered to a human.

The invention is further defined by the following paragraphs:

1. A compound selected from clozapine, norclozapine and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease in a subject wherein said compound causes mature B cells to be inhibited in said subject.

2. The compound for use according to paragraph 1 wherein the compound is clozapine or a pharmaceutically acceptable salt or solvates thereof.

3. The compound for use according to paragraph 1 or paragraph 2 wherein the mature B cells are class switched memory B cells.

4. The compound for use according to paragraph 1 or paragraph 2 wherein the mature B cells are plasmablasts.

5. The compound for use according to any one of paragraphs 1 to 4 wherein the pathogenic immunoglobulin driven B cell disease is a pathogenic IgG driven B cell disease.

6. The compound for use according to any one of paragraphs 1 to 5 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, autoimmune alopecia, vitiligo, dermatitis herpetiformis, chronic autoimmune urticaria, coeliac disease, Graves' disease, Hashimoto's thyroiditis, Type 1 diabetes mellitus, autoimmune Addison's disease, autoimmune haemolytic anaemia, autoimmune thrombocytopenic purpura, cryoglobulinemia, pernicious anaemia, myasthenia gravis, multiple sclerosis, neuromyelitis optica, autoimmune epilepsy and encephalitis, autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis.

7. The compound for use according to paragraph 6 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid.

8. The compound for use according to any one of paragraphs 1 to 4 wherein the pathogenic immunoglobulin driven B cell disease is a pathogenic IgA driven B cell disease.

9. The compound for use according to any one of paragraphs 1 to 5 and 8 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of dermatitis herpetiformis, linear IgA disease, coeliac disease, IgA nephropathy, pemphigus vulgaris, pemphigus foliaceus, cicatricial pemphigoid and bullous pemphigoid.

10. The compound for use according to paragraph 9 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of dermatitis herpetiformis and linear IgA disease. 11. A pharmaceutical composition comprising a compound selected from clozapine, norclozapine and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof; and a

pharmaceutically acceptable diluent or carrier, for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease in a subject wherein said compound causes mature B cells to be inhibited in said subject.

12. The pharmaceutical composition for use according to paragraph 11 wherein the pharmaceutical composition is administered orally.

13. The pharmaceutical composition for use according to paragraph 11 or paragraph 12 wherein the mature B cells are class switched memory B cells or plasmablasts.

14. A compound selected from clozapine, norclozapine and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use according to any one of paragraphs 1 to 10 in combination with a second or further therapeutic agent for the treatment or prevention of a pathogenic immunoglobulin driven B cell disease.

15. The compound selected from clozapine, norclozapine and prodrugs thereof and

pharmaceutically acceptable salts and solvates thereof for use according to paragraph 14 wherein the second or further substance for the treatment or prevention of a pathogenic immunoglobulin driven B cell disease is selected from anti-TNFa agents (such as anti-TNFa antibodies e.g. infliximab or adalumumab), calcineurin inhibitors (such as tacrolimus or cyclosporine), antiproliferative agents (such as mycophenolate e.g. as mofetil or sodium, or azathioprine), general anti-inflammatories (such as hydroxychloroquine or NSAIDS such as ketoprofen and colchicine), mTOR inhibitors (such as sirolimus), steroids (such as prednisone), anti-CD80/CD86 agents (such as abatacept), anti-CD-20 agents (such as anti-CD-20 antibodies e.g. rituximab). anti- BAFF agents (such as anti- BAFF antibodies e.g. tabalumab or belimumab, or atacicept), immunosuppressants (such as methotrexate or cyclophosphamide), anti-FcRn agents (e.g. anti-FcRn antibodies) and other antibodies (such as ARGX-113, PRN-1008, SYNT-001, veltuzumab, ocrelizumab, ofatumumab, obinutuzumab, ublituximab, alemtuzumab, milatuzumab, epratuzumab and blinatumomab).

16. A method of treatment or prevention of a pathogenic immunoglobulin driven B cell disease in a subject by administering to said subject an effective amount of a compound selected from clozapine, norclozapine and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof, in particular, wherein said compound causes mature B cells to be inhibited in said subject.

17. A method according to paragraph 16 wherein the compound is clozapine or a pharmaceutically acceptable salt or solvates thereof. 18. A method according to paragraph 16 or paragraph 17 wherein the pathogenic immunoglobulin driven B cell disease is a pathogenic IgG driven B cell disease.

19. A method according to any one of paragraphs 16 to 18 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, autoimmune alopecia, vitiligo, dermatitis herpetiformis, chronic autoimmune urticaria, coeliac disease, Graves' disease,

Hashimoto's thyroiditis, Type 1 diabetes mellitus, autoimmune Addison's disease, autoimmune haemolytic anaemia, autoimmune thrombocytopenic purpura, cryoglobulinemia, pernicious anaemia, myasthenia gravis, multiple sclerosis, neuromyelitis optica, autoimmune epilepsy and encephalitis, autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis.

20. The compound for use according to paragraph 19 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of pemphigus vulgaris, pemphigus foliaceus and bullous pemphigoid.

21. The compound for use according to paragraph 16 or paragraph 17 wherein the pathogenic immunoglobulin driven B cell disease is a pathogenic IgA driven B cell disease.

22. The compound for use according to any one of paragraphs 16, 17 and 21 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of dermatitis herpetiformis, linear IgA disease, coeliac disease, IgA nephropathy, pemphigus vulgaris, pemphigus foliaceus, cicatricial pemphigoid and bullous pemphigoid.

23. The compound for use according to paragraph 22 wherein the pathogenic immunoglobulin driven B cell disease is a disease selected from the group consisting of dermatitis herpetiformis and linear IgA disease.

The invention is further defined by the following clauses:

1. A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component in a subject wherein said compound causes mature B cells to be inhibited in said subject.

2. A method of treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component in a subject by administering to said subject an effective amount of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof wherein said compound causes mature B cells to be inhibited in said subject.

3. Use of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof in the manufacture of a medicament for the treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component in a subject wherein said compound causes mature B cells to be inhibited in said subject.

4. The compound for use, method or use according to any one of clauses 1 to 3 wherein the compound is pegylated clozapine or a pharmaceutically acceptable salt or solvate thereof.

5. The compound for use, method or use according to any one of clauses 1 to 4 wherein the mature B cells are class switched memory B cells.

6. The compound for use, method or use according to any one of clauses 1 to 4 wherein the mature B cells are plasmablasts.

7. The compound for use, method or use according to any one of clauses 1 to 6 wherein the pathogenic immunoglobulin driven B cell disease with a T cell component is a disease selected from the group consisting of vitiligo, psoriasis, coeliac disease, dermatitis herpetiformis, discoid lupus erythematosus, dermatomyositis, polymyositis, Type 1 diabetes mellitus, autoimmune Addison's disease, multiple sclerosis, interstitial lung disease, Crohn's disease, ulcerative colitis, thyroid autoimmune disease, autoimmune uveitis, primary biliary cirrhosis, primary sclerosing cholangitis, undifferentiated connective tissue disease, autoimmune thrombocytopenic purpura, mixed connective tissue disease, an immune-mediated inflammatory disease (IMID) such as scleroderma, rheumatoid arthritis, Sjogren's disease, and an autoimmune connective tissue disease such as systemic lupus erythematosus.

8. The compound for use, method or use according to clause 7 wherein the pathogenic

immunoglobulin driven B cell disease with a T cell component is psoriasis, a connective tissue disease such as systemic lupus erythematosus, or an immune-mediated inflammatory disease (IMID) such as scleroderma, rheumatoid arthritis or Sjogren's disease.

9. The compound for use, method or use according to any one of clauses 1 to 6 wherein the pathogenic immunoglobulin driven B cell disease with a T cell component is graft versus host disease. 10. The compound for use, method or use according to any one of clauses 1 to 9 wherein the compound has the effect of decreasing CD19 (+) B cells and/or (-) B-plasma cells.

11. A pharmaceutical composition comprising a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof; and a pharmaceutically acceptable diluent or carrier, for use in the treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component in a subject wherein said compound causes mature B cells to be inhibited in said subject.

12. The pharmaceutical composition for use according to clause 11 wherein the pharmaceutical composition is administered orally.

13. The pharmaceutical composition for use according to either clause 11 or 12 wherein the pharmaceutical composition is formulated as a liquid or solid, such as a syrup, suspension, emulsion, tablets, capsule or lozenge.

14. The pharmaceutical composition for use according to any one of clauses 11 to 14 wherein the mature B cells are class switched memory B cells.

15. The pharmaceutical composition for use according to any one of clauses 11 to 14 wherein the mature B cells are plasmablasts.

16. A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use according to any one of clauses 1 and 6 to 10 in combination with a second or further therapeutic agent for the treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component.

17. The compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use according to clause 16 wherein the second or further substance for the treatment or prevention of a pathogenic immunoglobulin driven B cell disease with a T cell component is selected from anti-TNFa agents (such as anti-TNFa antibodies e.g. infliximab or adalumumab), calcineurin inhibitors (such as tacrolimus or cyclosporine), antiproliferative agents (such as mycophenolate e.g. as mofetil or sodium, or azathioprine), general anti-inflammatories (such as hydroxychloroquine or NSAIDS such as ketoprofen and colchicine), mTOR inhibitors (such as sirolimus), steroids (such as prednisone), anti-CD80/CD86 agents (such as abatacept), anti-CD-20 agents (such as anti-CD-20 antibodies e.g. rituximab), anti- BAFF agents (such as anti- BAFF antibodies e.g. tabalumab or belimumab, or atacicept), immunosuppressants (such as methotrexate or cyclophosphamide), anti-FcRn agents (e.g. anti-FcRn antibodies) and other antibodies (such as ARGX-113, PRN-1008, SYNT-001, veltuzumab, ocrelizumab, ofatumumab, obinutuzumab, ublituximab, alemtuzumab, milatuzumab, epratuzumab and blinatumomab).

The invention is further defined by the following aspects:

1. A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use in the treatment or prevention of a pathogenic IgE driven B cell disease in a subject wherein said compound causes mature B cells to be inhibited in said subject.

2. A method of treatment or prevention of a pathogenic IgE driven B cell disease in a subject by administering to said subject an effective amount of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof wherein said compound causes mature B cells to be inhibited in said subject.

3. Use of a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof in the manufacture of a medicament for the treatment or prevention of a pathogenic IgE driven B cell disease in a subject wherein said compound causes mature B cells to be inhibited in said subject.

4. The compound for use, method or use according to any one of aspects 1 to 3 wherein the compound is pegylated clozapine or a pharmaceutically acceptable salt or solvate thereof.

5. The compound for use, method or use according to any one of aspects 1 to 4 wherein the mature B cells are class switched memory B-cells.

6. The compound for use, method or use according to any one of aspects 1 to 4 wherein the mature B cells are plasmablasts.

7. The compound for use, method or use according to any one of aspects 1 to 6 wherein the pathogenic IgE driven B cell disease is a disease selected from the group consisting of atopic asthma, atopic dermatitis, chronic non-autoimmune urticaria, Churg-Strauss vasculitis, allergic rhinitis and allergic eye disease preferably atopic dermatitis, atopic asthma, allergic rhinitis and eosinophilic esophagitis.

8. The compound for use, method or use according to any one of aspects 1 to 7 wherein the compound has the effect of decreasing CD19 (+) B cells and/or (-) B plasma cells.

9. A pharmaceutical composition comprising a compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof; and a pharmaceutically acceptable diluent or carrier, for use in the treatment or prevention of a pathogenic IgE driven B cell disease in a subject wherein said compound causes mature B cells to be inhibited in said subject.

10. The pharmaceutical composition for use according to aspect 9 wherein the pharmaceutical composition is administered orally.

11. The pharmaceutical composition for use according to either aspect 9 or 10 wherein the pharmaceutical composition is formulated as a liquid or solid, such as a syrup, suspension, emulsion, tablets, capsule or lozenge.

12. The pharmaceutical composition for use according to any one of aspects 9 to 11 wherein the mature B-cells are class switched memory B cells.

13. The pharmaceutical composition for use according to any one of aspects 9 to 11 wherein the mature B-cells are plasmablasts.

14. A compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use according to any one of aspects 1 and 4 to 8 in combination with a second or further therapeutic agent for the treatment or prevention of a pathogenic IgE driven B cell disease.

15. The compound selected from clozapine, norclozapine, pegylated derivatives thereof and prodrugs thereof and pharmaceutically acceptable salts and solvates thereof for use according to aspect 14 wherein the second or further substance for the treatment or prevention of a pathogenic IgE driven B cell disease is selected from anti-TNFa agents (such as anti-TNFa antibodies e.g.

infliximab or adalumumab), calcineurin inhibitors (such as tacrolimus or cyclosporine),

antiproliferative agents (such as mycophenolate e.g. as mofetil or sodium, or azathioprine), general anti-inflammatories (such as hydroxychloroquine or NSAIDS such as ketoprofen and colchicine), mTOR inhibitors (such as sirolimus), steroids (such as prednisone), anti-CD80/CD86 agents (such as abatacept), anti-CD-20 agents (such as anti-CD-20 antibodies e.g. rituximab). anti- BAFF agents (such as anti- BAFF antibodies e.g. tabalumab or belimumab, or atacicept), immunosuppressants (such as methotrexate or cyclophosphamide), anti-FcRn agents (e.g. anti-FcRn antibodies) and other antibodies (such as ARGX-113, PRN-1008, SYNT-001, veltuzumab, ocrelizumab, of atumumab, obinutuzumab, ublituximab, alemtuzumab, milatuzumab, epratuzumab and blinatumomab).

Examples

Example 1 First Observational Study on human patients on anti-psychotic therapy

To assess a possible association between antibody deficiency and clozapine use the inventors undertook a cross-sectional case control study to compare the immunoglobulin levels and specific antibody levels (against Haemophilus B (Hib), Tetanus and Pneumococcus) in patients taking either clozapine or alternative antipsychotics.

Method

Adults (>18yrs) receiving either clozapine or non-clozapine antipsychotics were recruited during routine clinic visits to ten Community Mental Health Trust (CMHT) outpatient clinics in Cardiff & Vale and Cwm Taf Health Boards by specialist research officers between November 2013 and December 2016 (Table 1). Following consent, participants completed a short lifestyle, drug history and infection questionnaire followed by blood sampling. Where required, drug histories were confirmed with the patient's General Practice records. Formal psychiatric diagnoses and antipsychotic medication use were confirmed using the medical notes, in line with other studies. Patients' admission rates were confirmed by electronic review for the 12-month period prior to recruitment. Patients with known possible causes of hypogammaglobulinemia including prior chemotherapy, carbamazepine, phenytoin, antimalarial agents, captopril, high-dose glucocorticoids, hematological malignancy and 22qll deletion syndrome were excluded.

Clinical and immunological data from 13 patients taking clozapine, 11 of whom had been referred independently of the study for assessment in Immunology clinic, are presented in Table 3.

Laboratory data on these, healthy controls and patients with common variable immunodeficiency (CVID) are shown in Figure 3. The 11 independently referred patients were excluded from the overall study analysis.

Immunoglobulin levels (IgG, IgA and IgM) were assayed by nephelometry (Siemens BN2

Nephelometer; Siemens), serum electrophoresis (Sebia Capillarys 2; Sebia, Norcross, GA, USA) and, where appropriate, serum immunofixation (Sebia Hydrasys; Sebia, Norcross, GA, USA). Specific antibody titres against Haemophilus influenzae, Tetanus and Pneumococcal capsular polysaccharide were determined by ELISA (The Binding Site, Birmingham, UK). Lymphocyte subsets, naive T cells and EUROclass B cell phenotyping were enumerated using a Beckman Coulter FC500 (Beckman Coulter, California, USA) flow cytometer. All testing was performed in the United Kingdom Accreditation Service (UKAS) accredited Immunology Laboratory at the University Hospital of Wales. Laboratory adult reference ranges for immunoglobulin levels used were, IgG 6-16g/L, IgA 0.8-4g/L, IgM 0.5-2g/L. Statistical analysis of the laboratory and clinical data was performed using Microsoft Excel and Graphpad Prism version 6.07 (Graphpad, San Diego, California, USA). Independent samples t-test were performed unless D'Agoustino & Pearson testing showed significant deviation from the Gaussian distribution, in which case the non-parametric Mann-Whitney test was used. All tests were two-tailed, using a significance level of p<0.05.

Results

Study Participants

A total of 291 patients taking clozapine and 280 clozapine-naive patients were approached and 123 clozapine and 111 clozapine-naive patients consented to the study (Table 1). Recruitment was stopped as per protocol when the target of 100 patients in each group had been achieved. There were small differences in gender with more males in the clozapine-treated group (53% versus 50%) and a lower mean age in the clozapine group (45 versus 50 years). These differences are unlikely to be relevant as there are no gender differences in the adult reference range for serum

immunoglobulins and there is a male predominance in schizophrenia. Levels of smoking, diabetes, COPD/asthma, and alcohol intake were similar between the groups. More patients were admitted to hospital with infection in the clozapine group (0.12 vs 0.06 per patient year) and more took >5 courses of antibiotics per year compared with controls (5.3% vs 2%). The possible impact of a diagnosis of schizophrenia, medications and smoking as risk factors for antibody deficiency were assessed in a subgroup analysis (Table 2). Table 1 Clozapine-treated and clozapine-naive patient characteristics

Effects of clozapine on antibody levels

Figure 1 A-C shows significantly reduced concentrations of all three immunoglobulin classes (IgG, IgA and IgM) in patients receiving clozapine, with a shift towards lower immunoglobulin levels in the distribution as a whole for each of IgG, IgA and IgM compared to the clozapine-naive control group. The percentages of the 123 patients having immunoglobulin levels below the reference range were IgG 9.8% (p<0.0001), IgA 13.0% (p<0.0001) and IgM 38.2% (p<0.0001) compared with the 111 clozapine-naive IgG 1.8%, IgA 0.0% and IgM 14.4%. Large percentages of both clozapine-treated and clozapine-naive patients had specific antibody levels below the protective levels for HiB (51% and 56% less than 1 mcg/ml, (Orange et al., 2012)), Pneumococcus (54% and 56% less than 50mg/L,

(Chua et al., 2011)) and Tetanus (12% and 14% less than O.llU/ml). The Pneumococcal IgA (31U/ml vs 58.4U/ml p< 0.001) and IgM (58.5U/ml vs 85.0U/ml p<0.001) levels are significantly lower in clozapine-treated versus clozapine-naive patients.

Subgroup analysis (Table 2) was undertaken to determine if the reductions in immunoglobulins were potentially explained by confounding factors including any other drugs, a diagnosis of schizophrenia and smoking. The assessment of the effect of excluding other secondary causes of antibody deficiency (plus small numbers where additional diagnoses were uncovered - Table 1) is shown in Column B. The number of patients excluded on the basis of taking anti-epileptic medications was higher in the clozapine-treated group and is likely to reflect the use of these agents for their mood stabilizing properties rather than as treatment for epilepsy.

Table 2 Immunoglobulin levels and specific antibody levels in sub-groups A-D

Data shown as mean ± 1 SEM unless otherwise stated. * Independent T test (normally distributed) or ^† Mann-Whitney (non-normally distributed)

Levels of significance: */† p<0.05, **/†† p<0.005, ***/††† p<0.0005, ****/†††† p<0.0001

The association of clozapine with reduced IgG, IgA, IgM and Pneumococcal IgA and IgM remained statistically significant in all subgroups with 95% confidence intervals including when psychiatric diagnoses were restricted to schizophrenia only (Column C), and when non-smokers were excluded (Column D). When secondary causes of antibody deficiency were excluded (Column B) the odds ratios (with 95% confidence interval) for reduced immunoglobulins were IgG 9.02 (1.11 - 73.7), IgA: 32.6 (1.91 - 558) and IgM: 2.86 (1.42 - 5.73). In addition, a longer duration of clozapine therapy is associated with lower serum IgG levels (p 0.014) shown in Figure 2. This is not observed in clozapine- naive patients treated with alternative antipsychotic drugs, despite a longer treatment duration than the clozapine therapy group.

Immunological assessment of referred patients taking clozapine

Thirteen patients on clozapine were independently referred for assessment of antibody deficiency to Immunology clinic. Two had previously been recruited to the study and the eleven others are not included in the study to avoid bias. Five of the thirteen patients had been identified through the all Wales calculated globulin screening program. It was thus possible to undertake a more detailed immunological assessment in this group of thirteen 'real life' patients to provide additional background information (Table 3). Table 3 Immunological characteristics of the 13 referred clozapine patients

Certain additional analysis shown in Figures ID, 3B, 4B and 5 was done on a slightly different set of referred clozapine patients comprising the 13 referred to in Table 3, plus 4 additionally recruited patients. In respect of Figure ID, 4 of the 17 patients were removed for various reasons therefore the number of patients for which data is presented is 13. In respect of Figure 3B, the number of patients for which data is presented is shown in the Figure. In respect of Figure 4B, the number of patients for which data is presented is stated below. In respect of Figure 5, the number of patients for which data is presented is 15.

Immunoglobulins were reduced in all patients (mean IgG 3.6g/L, IgA 0.34g/L and IgM 0.21g/L). There was no severe overall lymphopenia or B cell lymphopenia, however, all patients had a major reduction in the percentage of CSMB (mean 1.87%, reference range 6.5-29.1%). A substantial reduction of CSMB is characteristic of patients with common variable immunodeficiency (CVID), the commonest severe primary immunodeficiency in adults. The percentages of CSMB in these clozapine-treated and CVID patients compared to healthy controls are shown in Figure 3A

(p<0.0001). The plasmablast levels for 6 of the clozapine patients compared to CVID patients and healthy controls are shown in Figure 4A (p=0.04) and in Figure 3B with age matched CVID and healthy controls. A reduction of plasmablasts is also characteristic of patients with common variable immunodeficiency (CVID) and this was also observed in clozapine treated patients. Responses to vaccination were impaired in 10/11 patients assessed and management included emergency backup antibiotics for 2/13 patients, prophylactic antibiotics in 9/13 and 6/13 patients were treated with immunoglobulin replacement therapy (IGRT). No patients discontinued clozapine because of antibody deficiency. The inflammatory or granulomatous complications which occur in a subset of CVID patients were not observed.

Vaccine specific-lgG responses are routinely evaluated as part of clinical assessment and summarised in Figure 4B. At initial assessment, levels below putative protective threshold were common with IgG to Flaemophilus influenza B (HiB) < lmcg/ml in 12/16 patients (75%); Pneumococcus-lgG < 50mg/L in 15/16 patients (94%); and Tetanus-lgG < 0.1 lU/mL in 6/16 patients (38%) individuals tested. Post- Menitorix (FliB/MenC) vaccination serology was assessed after 4 weeks, with 5/12 (42%) individuals failing to mount a Haemophilus-lgG response >lmcg/ml, and 1/12 failing to exceed the >0.1 lU/mL post-vaccination Tetanus-lgG level defined by the World Health Organisation. Following Pneumovax II, 8/11 (73%) individuals failed to develop an IgG response above a threshold of >50mg/L.

Figure 5 shows a gradual recovery in terms of the serum IgG level from 3.5g/L to 5.95g/L over 3 years but without clear improvement in IgA or IgM following cessation of clozapine.

One patient subsequently discontinued clozapine because of neutropenia which normalized on clozapine cessation. Over the following 24 months the serum IgG level gradually increased from 3.3g/L to 4.8g/L and then 5.95g/L while IgA and IgM remained low. The increase in IgG was accompanied by a concomitant increase in class switched memory B cells from 1.58 - 2.77%, suggesting a gradual recovery on withdrawal of clozapine. Figure ID shows a density plot showing distribution of serum immunoglobulin levels in patients receiving clozapine referred for Immunology assessment. Serum immunoglobulin distributions for clozapine-treated (n = 94) and clozapine-naive (n = 98) are also shown for comparison- adapted from (Ponsford et al 2018a). Dotted lines represent the 5 ^th and 95 ^th percentiles for healthy adults. A leftward shift (reduction) in the distribution curves of total immunoglobulin is observed in patients on clozapine for each of IgG, IgA and IgM compared to clozapine naive patients; this finding was particularly marked for the additionally recruited clozapine referred patients.

Summary of results

Clozapine treatment in patients led to a significant reduction of all immunoglobulin types.

Percentages of patients below the immunoglobulin reference ranges were higher in clozapine treated (n=123) as compared with clozapine naive patients (n=lll) (IgG <6g/L: 9.8% vs 1.8%; IgA <0.8g/L: 13.1% vs 0..0%; IgM <0.5g/L: 38.2% vs 14.2%) (p<0.0001) (see Figure 1 A-C).

Extending the duration of clozapine treatment was associated with progressively reduced IgG levels in patients treated with clozapine but not in clozapine naive patients who were on other antipsychotic medication (see Figure 2).

Notably the effect of clozapine on IgG levels was seen to be reversible, albeit slowly (years), consistent with an impact of clozapine on long-lived lgG+ plasma cells in particular.

Specific IgG antibodies were below protective levels in both clozapine-treated and clozapine-naive groups (HiB 51.2% vs 55.9%; Pneumococcal 53.7% vs 55.9%; Tetanus 12.2% vs 13.5%)). Flowever, pneumococcal IgA and IgM levels were significantly lower in clozapine-treated patients as compared with clozapine-naive patients (IgA 31.0 U/L vs 58.4 U/L; IgM 58.5 U/L vs 85 U/L) (p<0.001) (see Table 2).

Mean levels of CSMBs were significantly reduced at 1.87% in clozapine-treated patients referred independently to clinic and not included in the overall study (n=12) and in CVID patients (n=54) as compared with healthy controls (n=36) and the reference range of 6.5-29.1% (p<0.0001) (see Figure 3A). Mean levels of plasmablasts were also reduced in clozapine-treated patients (p=0.04).

Figure 3B shows an extension of the data in Figure 3A in which referred clozapine patients are compared to age matched CVID and health control subjects. The first graph shows that total B cell numbers are similar between clozapine, CVID and healthy controls and the second graph demonstrates no significant difference between clozapine treated and healthy control marginal zone B cell numbers while there is an increased number observed in CVID patients. The lower two graphs show a significant reduction in both CSMB and plasmablasts in both clozapine treated and CVID patients over healthy controls. Example 2

Second Observational Study on human patients on anti-psychotic therapy

Using a cross-sectional observational design in patients on anti-psychotic therapy, this study sought to test the association between clozapine use, immunophenotype - specifically circulating B cell subsets and immunoglobulin levels - and documented infections, in comparison to other anti psychotic medication. The study recruited patients established on clozapine and those on other antipsychotic drugs from Ashworth Hospital and outpatients from community mental health services in Mersey Care NHS Foundation Trust. The findings partly provide validation of those from a published observational study (Ponsford et al., 2018a) in an orthogonal population, in addition to extending insights into the impact of clozapine on B cell populations through more detailed immunophenotypic analysis.

The study entailed a single blood test for detailed immunological analysis and completion of a clinical research form-based questionnaire detailing important clinical parameters including documented infection history, past medical history and concurrent medication use. The analysis aims to identify any association between clozapine, circulating B cell levels/function and immunoglobulin levels, its frequency and severity, as well as specificity in relation to other antipsychotic medications.

Study Aims and Objectives

The specific research questions this study sought to answer are:

Primary Outcomes: i) Is chronic treatment with clozapine associated with (a) a higher proportion of those with specific B cell subsets (namely class-switched memory B cells and plasma cells) below reference ranges and (b) a higher proportion of those with circulating immunoglobulin levels (IgG, IgA and IgM) below references compared to proportions below reference range observed in controls?

Secondary Outcomes: ii) Is clozapine associated with reductions in specific antibodies (e.g. pneumococcus, tetanus and Hib) compared to controls? iii) Is clozapine use associated with an effect on circulating T cells (number/function) compared to controls? iv) Is clozapine associated with a higher frequency of infections and antibiotic use than controls? v) Are the primary outcomes related to duration of clozapine therapy? Immune Biomarkers

The following immune biomarkers were tested:

1. Total IgG, IgM, IgA, and serum electrophoresis with immunofixation if appropriate;

2. Specific IgG levels - tetanus toxoid, pneumococcus, Hib (± IgA and IgM for pneumococcus);

3. Detailed immune cell phenotyping through FACS analysis, including: a. Lymphocyte phenotypes - (including CD3, CD4, CD8, CD19, CD56) b. B cell panel (based on the EUROCIass classification of B cell phenotype (Wehr et al., 2008)) which includes CSMB cells and plasmablasts c. Naive T cell panel

All immune biomarker samples are processed and analysed in a UKAS Accredited validated NHS laboratory.

Results

The major findings from the immunophenotypic analysis are detailed below: a. Significantly reduced levels of each of circulating total IgG, IgA and IgM in patients on clozapine versus patients who have never taken clozapine (i.e. control, clozapine naive) (see Figure 6A-C). These reductions are relatively greater for Ig of the A and M subclass. In addition, a significantly lower specific IgG antibody against pneumococcus is present in those treated with clozapine (see Figure 7). b. Overall CD19 ⁺ B cell numbers (both absolute and when expressed as a proportion of total circulating B cells, %B) are not significantly different between groups (see Figure 8A-B). c. No statistically significant difference in the number of naive (CD19 ⁺ CD27 ) B cells (see Figure 9A- C). d. Statistical trend to a specific reduction in class-switched memory B cells (P= 0.08 vs control, CD27 ⁺ IgM IgD as %B) in those treated with clozapine (see Figure 11A-C) without perturbation of the overall memory B cell pool (see Figure 10A-C) or lgM ^hl IgD ¹⁰ memory B cell subpopulation (see Figure 12A-C). e. No significant difference between groups in circulating levels of transitional B cells or marginal zone (MZ) B cells (See Figures 13A-C and 14A-C). f. Significant reduction in levels of plasmablasts in patients treated with clozapine vs control clozapine naive expressed as percentage of total circulating B cells (%B) or total lymphocytes (%L) (see Figure 15A-C).

Example 3

In vivo wild type mouse study - effect of clozapine versus haloperidol

The impact of clozapine on B cell development, differentiation and function (inferred from circulating immunoglobulin levels) in primary (bone marrow) and secondary (spleen and also mesenteric lymph node) lymphoid tissue in wild type mice in the steady state (i.e. in the absence of specific immunological challenge) was assessed.

The specific objectives were to: a) Determine the impact of clozapine on major B cell subsets in bone marrow and key secondary lymphoid organs (spleen and mesenteric lymph node) of healthy mice. b) Define whether a dose-response relationship exists for clozapine on aspects of the B cell immunophenotype. c) Assess the effect of clozapine administration on the circulating immunoglobulin profile of healthy mice. d) Determine the specificity of clozapine's effect on the above readouts by comparison to another antipsychotic agent.

Method

Animals:

Young adult (age 7-8 weeks) C57BL/6 mature female mice were used for the study. Mice were housed at 22°C in individually ventilated cages with free access to food and water and a 12-h light/dark cycle (8 a.m./8 p.m.). Mice acclimatised for 1 week on arrival prior to initiating experiments.

Experimental groups and dose selection:

Mice were allocated into one of five experimental groups as follows:

1. Control saline

2. Clozapine low dose 2.5 mg/kg

3. Clozapine intermediate dose 5 mg/kg

4. Clozapine high dose 10 mg/kg 5. Haloperidol 1 mg/kg (intermediate dose)

Dosing was given in staggered batches with each batch containing mice assigned to each experimental arm to reduce bias.

Dose selection was initially based on a literature review of studies administering these drugs chronically to mice (Ishisaka et al., 2015; Li et al., 2016a; Mutlu et al., 2012; Sacchi et al 2017;

Simon et al., 2000; Tanyeri et al., 2017), the great majority of which had employed the

intraperitoneal (IP) route of administration: clozapine (1.5, 5, 10, 25 mg/kg/day) (Gray et al., 2009; Moreno et al., 2013); haloperidol (0.25 mg/kg, 1 mg/kg/day) (Gray et al., 2009) and taking into account the LD50 for both drugs (clozapine 200 mg/kg, haloperidol 30 mg/kg).

Subsequently, pilot studies were undertaken to assess the impact of these, particularly of the higher doses of clozapine, to refine dose selection and maximise the welfare of treated mice. Clear dose- related sedative effects were evident from dosages of clozapine starting at 5 mg/kg, with marked psychomotor suppression (with respect to depth and duration) observed at the highest doses assessed (20 mg/kg and 25 mg/kg). In addition, effects on thermoregulation were also evident, necessitating use of a warming chamber and general supportive measures to defend thermal homeostasis. These adverse effects were consistent with the known (on-target) profile of clozapine in preclinical (Joshi et al., 2017; McOmish et al., 2012; Millan et al., 1995; Williams et al., 2012) and clinical settings (Marinkovic et al., 1994), with tolerance developing after the initial few days of dosing, as has been described in humans (Marinkovic et al., 1994).

Mice (n=12/group) were treated by once daily IP injection of the respective control

solution/clozapine/haloperidol for 21 consecutive days. Biological samples for immunophenotyping:

At the end of the experimental period, mice were humanely euthanised and blood samples obtained for serum separation, storage at -80°C and subsequent measurement of immunoglobulin profiles (including the major immunoglobulin subsets IgGl, lgG2a, lgG2b, lgG3, IgA, IgM, and both light chains kappa and lambda) by ELISA.

In parallel, tissue samples were rapidly collected from bone marrow (from femur), spleen and mesenteric lymph nodes for evaluation of cellular composition across these compartments using multi-laser flow cytometric detection and analysis.

B cell immunophenotyping by flow cytometry:

Focused B cell FACS (fluorescence-activated cell sorter) panels were prepared separately for both primary (bone marrow) and secondary (spleen/lymph node) lymphoid tissue to allow an evaluation of drug impact on the relative composition of B cell subsets spanning the spectrum of antigen- independent and -dependent phases of B cell development.

Individual antibodies employed for flow cytometry panels were pilot tested in the relevant tissues (i.e. bone marrow, spleen and mesenteric lymph node) and the optimal dilution of each antibody determined to enable clear identification of subpopulations. FACS data were extracted by BD FACSymphony and analysed by FlowJo software.

Results

Body weight:

Clozapine (CLZ) induced a transient fall in body weight at both 5 mg/kg and 10 mg/kg doses, maximal by 3 days but recovering fully to baseline by day 9 with progressive weight gain beyond this (see Figures 16 and 17). This finding is likely to reflect the sedative effect of clozapine on fluid/food intake during the initial few days of dosing, with evidence of tolerance to this emerging over the course of the experiment.

Early B cell development in bone marrow:

B cells originate from hematopoietic stem cells (FISCs), multipotent cells with self-renewal ability, located in the bone marrow. This early B cell development occurs from committed common lymphoid progenitor cells and progresses through a set of stages, dependent on physical and soluble chemokine/cytokine interactions with bone marrow stromal cells, defined using cell surface markers. The earliest B cell progenitor is the pre-pro-B cell, which expresses B220 and has germline Ig genes. Next, pro-B cells rearrange their H (heavy) chain Igp genes, and express CD19 under the control of transcription factor Pax5. At the pre-B cell stage, cells downregulate CD43, express intracellular Igp, and then rearrange the L (light) chain and upregulate CD25 in an Irf4-dependent manner.

Successfully selected cells become immature (surface lgM ⁺ lgD ) B cells. Immature B cells are tested for autoreactivity through a process of central tolerance and those without strong reactivity to self antigens exit the bone marrow via sinusoids to continue their maturation in the spleen.

No overall reduction in B cells in the bone marrow (BM) was observed at any dose of clozapine (see Figure 18). However, a significant increase in the proportion of very early B cell progenitors, the pre- pro B cells (i.e. B220 ⁺ CD19 CD43 ⁺ CD24 ^lo BP- igM lgD ) was observed with 10 mg/kg clozapine, without any change evident in the subsequent pro-B cell fraction (see Figure 18). In contrast, no significant effect of haloperidol was evident on any of these early developing B cell subsets.

Examination of subsequent stages of B cell development in bone marrow revealed a reduction in pre-B cells (i.e. B220 ⁺ CD19 ⁺ CD43 CD24 ⁺ BP-TlgM lgD ) in mice treated with clozapine (see Figure 19). Notably this effect exhibited dose-dependency, with a significant difference observed verses control mice with even the lowest dose of clozapine employed (2.5 mg/kg). Furthermore, the percentage of pre-B cells that were proliferating (i.e. B220 ⁺ CD19 ⁺ CD43 CD24 ^hl BP-l ⁺ lgM lgD ) was diminished with clozapine, reaching significance for the 5 mg/kg dose (see Figure 19). Correspondingly, a reduction in the percentage of immature B cells in bone marrow was identified (i.e. B220 ⁺ CD19 ⁺ CD43

CD24lgMlgD ) (see Figure 19).

Together, these findings suggest a specific impact of clozapine on early B cell development, with a modest arrest between the pre-pro-B cell and pre-B cell stages in the absence of specific immunological challenge.

Peripheral B cell development - total splenic B cells:

After emigrating from the bone marrow, functionally immature B cells undergo further development in secondary lymphoid organs, enabling further exposure to (peripheral) self-antigen and peripheral tolerance (resulting in cell deletion through apoptosis, anergy or survival). The majority of immature B cells exiting bone marrow do not survive to become fully mature B cells, a process regulated by maturation and survival signals received in lymphoid follicles, including BAFF (B cell activating factor) secreted by follicular dendritic cells.

Mice treated with clozapine at 5 mg/kg and 10 mg/kg were seen to have a significantly lower percentage of splenic B cells (i.e. 6220^08- ) expressed as a proportion of total live splenocytes (see Figure 21). No effect was identified on other cell populations (i.e. B220TCR- ), which may include gd T cells (which do not express the ab T cell receptor, TCR), natural killer (NK) cells, or other rare lymphoid cell populations (see Figure 21). This was accompanied by a reciprocal increase in the percentage of splenic T cells (i.e. B220-TCR- +) (see Figure 21). In contrast, activated T cells (i.e. B220 ⁺ TCR- ⁺ ), reflecting a small proportion of total live splenocytes were reduced in dose- dependent fashion by clozapine compared to control, an effect also modestly apparent for haloperidol (see Figure 21).

These findings suggest that clozapine, but not haloperidol, is able to affect peripheral (splenic) B cells in addition to the observed changes in bone marrow B cell precursors.

Splenic B cell subpopulations:

Immature B cells exiting the bone marrow and entering the circulation are known as transitional B cells. These immature cells enter the spleen and competitively access splenic follicles to differentiate via transitional stages to immunocompetent naive mature B cells. This occurs sequentially in the follicle from transitional type 1 (Tl) cells, similar to immature B cells in bone marrow, to type 2 (T2) precursors. The latter are thought to be the immediate precursor of mature naive B cells. T2 B cells have been demonstrated to show greater potency in response to B cell receptor stimulation than Tl B cells, suggesting that the T2 subset may preferentially undergo positive selection and progression into the long-lived mature B cell pool (Petro et al., 2002).

Transitional cells can differentiate into follicular B cells, representing the majority of peripheral B cells residing in secondary lymphoid organs, or a less numerous population, marginal zone (MZ) B cells residing at the white/red pulp interface which are able to respond rapidly to blood-borne antigens/pathogens.

Mice treated with clozapine were found to have a mildly reduced proportion of newly emigrated transitional stage 1 (Tl) B cells in the spleen, including at the 2.5 mg/kg dose, which may in part reflect the reduction in percentage of bone marrow immature B cells (see Figure 22). In contrast, a small increase in the proportion of T2 B cells was identified across all doses of clozapine (see Figure 22), consistent with enhanced positive selection of Tl B cell subsets for potential progression into the long-lived mature B cell pool.

While clozapine administration reduced the splenic B cell contribution to live splenocytes (see Figure 21), no specific reductions were identified in either splenic follicular (i.e. B220 ⁺ CD19 ⁺ CD21 ^mid CD23 ⁺ ) or marginal zone (i.e. B220 ⁺ CD19 ⁺ CD21 ⁺ CD23 ^Lo/ ) B cell subsets (see Figure 22), suggesting that in the immunologically unchallenged state, clozapine administration in mice results in a global reduction in splenic B cell populations. Germinal centres (GCs) are micro-anatomical structures which form over several days in B cell follicles of secondary lymphoid tissues in response to T cell-dependent antigenic (e.g. due to infection or immunisation) challenge (Meyer-Hermann et al., 2012). Within GCs, B cells undergo somatic hypermutation of their antibody variable regions, with subsequent testing of the mutated B cell receptors against antigens displayed by GC resident follicular dendritic cells. Through a process of antibody affinity maturation, mutated B cells which higher affinity to antigen are identified and expanded. In addition, class switch recombination of the immunoglobulin heavy chain locus of mature naive (lgM ⁺ lgD ⁺ ) B cells occurs before and during GC reactions, modifying antibody effector function but not its specificity or affinity for antigen. This results in isotype switching from IgM to other immunoglobulin classes (IgG, IgA or IgE) in response to antigen stimulation.

GCs are therefore sites of intense B cell proliferation and cell death, with outcomes including apoptosis, positive selection for a further round of somatic hypermutation (i.e. cyclic re-entry), or B cell differentiation into antibody secreting plasma cells and memory B cells (Suan et al., 2017). In the steady state, GC cells (i.e. B220+CD19+lgD-CD95+GL-7+) formed a very small proportion of total live B cells in the spleen, with no differences observed versus control or haloperidol in response to clozapine administration (see Figure 22).

Bone marrow antibody secreting cell populations:

Antibody secreting cells represent the end-stage differentiation of the B cell lineage and are widely distributed in health across primary and secondary lymphoid organs, the gastrointestinal tract and mucosa (Tellier and Nutt, 2018). These cells all derive from activated B cells (follicular, MZ or Bl). Plasmablasts, representing short-lived cycling cells, can be derived from extra-follicular

differentiation pathway in a primary response (producing relatively lower affinity antibody), as well as from memory B cells that have undergone affinity maturation in the GC (Tellier and Nutt, 2018).

Plasmablasts developing in GCs can leave the secondary lymphoid organ and home to the bone marrow. Here, only a small proportion are thought to be retained and establish themselves in dedicated micro-environmental survival niches to mature into long-lived plasma cells (Chu and Berek, 2013), a process thought to be regulated by docking onto mesenchymal reticular stromal cells (Zehentmeier et al., 2014) and requiring haematopoietic cells (e.g. eosinophils) (Chu et al., 2011a), the presence of B cell survival factors (e.g. APRIL and IL-6) (Belnoue et al., 2008) and hypoxic conditions (Nguyen et al., 2018).

In the healthy state, the bone marrow houses the majority of long-lived plasma cells. Clozapine at 5 and 10 mg/kg induced a significant reduction in the percentage of long-lived plasma cells in the bone marrow (i.e. B220 ^lo CD19 lgD lgM CD20 CD38 ⁺⁺ CD138 ⁺ ) by ~30% compared to control (see Figure 20). In contrast, no effect of haloperidol was seen on this specific B cell population (see Figure 20). No significant changes were detected in either class-switched memory B cells (i.e. B220 ⁺ CD19 ⁺ CD27 ⁺ lgD lgM CD20 ⁺ CD38 ^+/ ) or plasmablasts (i.e. B220 ^lo CD19 ⁺ CD27lgD lgM CD20 CD38 ⁺⁺ ) in the bone marrow with any treatment, however both these represent a very small proportion of total B cells in the bone marrow in the immunologically unchallenged steady state (see Figure 20).

These findings indicate that clozapine can exert a specific effect to reduce the proportion of long- lived plasma cells in the bone marrow, a population thought to be the major source of stable antigen-specific antibody titres in plasma involved in humoral immune protection and, in pathogenic states, stable autoantibody production.

Circulating immunoglobulin levels:

Clozapine administration at both 5 and 10 mg/kg resulted in a reduction in circulating IgA levels compared to control, an effect not observed with haloperidol (see Figure 24; P, positive control; N, negative control). No other isotype classes were affected under the experimental conditions used (see Figure 24).

Mesenteric lymph nodes:

Under the current experimental conditions, no significant differences were identified between any of the groups in lymphocyte subpopulations assessed in mesenteric lymph nodes (MLN) (see Figure 23).

Conclusion

This study investigated the potential for clozapine to influence the immunophenotype of wild type mice in the steady state, specifically B cell subpopulations, with functional impact inferred through circulating levels of immunoglobulins. The major findings of this study are that 3 weeks parenteral (I.P.) administration of clozapine: a) Increases the proportion of pre-pro-B cells while reducing the proportion of later-stage pre- B cells and immature B cells in the bone marrow. b) Reduces the proportion of live splenocytes that are B cells. c) Exerts subtle effects on developing B cells in the spleen, specifically transitional B cell populations in favouring a greater proportion of T2 type cells. d) Significantly reduces the proportion of long-lived plasma cells in the bone marrow. e) Impacts on circulating immunoglobulin levels, specifically lowering IgA. f) Results in a dose-dependent decrease in the proportion of activated T cells in spleen which, in contrast to all the above findings, was also observed with the dose of haloperidol used.

Taken together, these observations indicate that clozapine exerts complex effects on B cell maturation in vivo, not limited to the late stages of B cell differentiation or activation. Specifically, the findings suggest that clozapine can influence the maturation of early B cell precursors, with a partial arrest of antigen-independent B cell development in the bone marrow.

In parallel, clear effects of clozapine are identified on peripheral B cell subpopulations, with a notable impact on reducing the overall B cell proportion of live splenocytes, and on long-lived antibody secreting plasma cells in the bone marrow. An impact on antibody secreting cells is likely to underlie the observed significant reduction in circulating IgA, particularly striking given the otherwise immunologically unchallenged state of the mice.

Notably, the impact on B cell subpopulations was not observed with a comparator antipsychotic agent, haloperidol, consistent with specificity of action of clozapine on B cell maturation. While the current experiments do not enable a distinction between a direct or indirect effect of clozapine on bone marrow, peripheral and late B cell populations, taken together with findings from separate in vitro B cell proliferation assays, an indirect effect is deemed more likely. This may involve a variety of other myeloid, lymphoid (e.g. T follicular helper cells) and/or (mesenchymal) stromal supportive cells.

Example 4

Mouse collagen-induced arthritis (CIA) model study - effect of clozapine

The CIA model is a well-established experimental model of autoimmune disease. The inventors have employed the CIA model as a highly clinically relevant experimental system in which B cell-derived pathogenic immunoglobulin made in response to a sample specific antigen drives autoimmune pathology to explore the potential efficacy of clozapine and its associated cellular mechanisms.

Method

Animals:

Adult (age 13-15 weeks) DBA/1 male mice were purchased from Envigo (Horst, Netherlands). Mice were housed at a 21°C ± 2°C in individually ventilated cages with free access to food and water and a 12-h light/dark cycle (7 am/7 pm). Mice were acclimatised for 1 week on arrival prior to initiating experiments.

Experimental groups and dose selection: Mice were allocated into one of five experimental groups as follows:

1. Control saline

2. Clozapine 5 mg/kg treatment from day 15 after immunization

3. Clozapine 10 mg/kg treatment from day 15 after immunization

4. Clozapine 5 mg/kg treatment from day 1 after immunization

5. Clozapine 10 mg/kg treatment from day 1 after immunization

Mice (n=10/group) were treated by once daily IP injection of the respective control

solution/clozapine until day 10 after onset of clinical features of arthritis. All experiments were approved by the Clinical Medicine Animal Welfare and Ethical Review Body (AWERB) and by the UK Home Office.

Anti-arthritic effect of clozapine in vivo:

DBA/1 mice were immunised with bovine type II collagen in CFA and monitored daily for onset of arthritis. Clozapine was administered daily by intraperitoneal injection at doses of 5 mg/kg or 10 mg/kg. Controls received vehicle (saline) alone. Treatment of mice commenced in one experiment on day 1 after immunisation and in a second experiment on day 15 after immunisation. Clinical scores and paw-swelling were monitored for 10 days following onset of arthritis. A clinical scoring system was used as follows. Arthritis severity was scored by an experienced, non-blinded investigator as follows: 0 = normal, 1 = slight swelling and/or erythema, 2 = pronounced swelling, 3 = ankylosis. All four limbs were scored, giving a maximum possible score of 12 per animal.

At the end of the experimental period, mice were humanely euthanised and bled by cardiac puncture to obtain blood samples for serum separation, storage at -80°C and subsequent measurement of specific anti-collagen immunoglobulin (IgGl and lgG2a isotypes) by ELISA. In parallel, spleen and inguinal lymph nodes were harvested for evaluation of cellular composition across these compartments using multi-laser flow cytometric detection and analysis. Numbers of B cell subsets in spleen and lymph nodes were determined by FACS.

Statistical Analysis:

Data were analyzed by one-way ANOVA with Tukey's or Dunnett's multiple comparison test or two- way ANOVA with Tukey's multiple comparison test as appropriate. All calculations were made using GraphPad Prism software. A P value less than 0.05 was considered significant.

Results

Effect of Clozapine on onset, clinical score and paw-swelling: Treatment of mice with clozapine was significantly effective in delaying the onset of arthritis post immunisation (see Figures 25 and 26). In particular, treatment with both doses of clozapine from day 1 was extremely effective in delaying arthritis onset (see Figures 25 and 26).

Furthermore, treatment with both doses of clozapine reduced overall clinical score when administered on day 1 and, in the case of 10 mg/kg clozapine, also reduced swelling of the first affected paw (see Figure 27). Clozapine administration also reduced the total number of affected paws compared to vehicle control, an effect significant with dosing at D1 (see Figure 28).

Effect of Clozapine on peripheral B cell subsets:

Mice treated with clozapine at all doses and time points (i.e. 5 mg/kg or 10 mg/kg from day 1 or day 15) were seen to have a significantly lower percentage of B220 ⁺ B cells in lymph nodes (see Figure 29). In addition, clozapine administered at 10 mg/kg from day 1 also significantly reduced the proportion of B220 ⁺ B cells in spleen.

Under the experimental conditions employed, no significant effect of clozapine was observed on plasma cell numbers in lymph node, however a significant reduction in the proportion of plasma cells was identified in spleen at a dose of 10 mg/kg clozapine given on day 1, with nominally lower values for plasma cells as a proportion of live cells at every other dose/time evaluated compared to control (see Figure 30).

Strikingly significant reductions in lymph node follicular B cells (B220 ⁺ lgD Fas ⁺ GL7 ^hl ) were observed in mice treated with clozapine across all doses/both time points (see Figure 31). In addition, the level of GL7 expression on follicular B cells in lymph node were significantly decreased across all clozapine treatment groups compared to vehicle treated controls (see Figure 32). There was evidence of dose- and time-dependency of effect with particularly profound reductions in GL7 expression in mice treated with clozapine from day 1 (see Figure 32).

Effect of Clozapine on anti-type II collagen IgG isotypes:

Clozapine administration at 5 or 10 mg/kg from day 1 or day 15 had no significant impact on serum lgG2a measured at a single time point. Flowever, clozapine administration led to nominal reductions in levels of IgGl across all doses tested, reaching statistical significance for the group treated with 10 mg/kg from day 15 (see Figure 33).

Effect of Clozapine on T follicular helper cells:

Treatment of mice with 5 mg/kg or 10 mg/kg of clozapine from day 1 or day 15 did not significantly affect proportions of CD4 ⁺ PD1 ⁺ CXCR5 ⁺ T follicular helper cells in lymph node or spleen (see Figure 34). Flowever, analysis of mean fluorescence intensity (MFI) revealed robust reductions in expression of PD-1 and CXCR5 on T follicular helper cells in mice-treated with clozapine (see Figures 35 and 36). Reduced expression of PD-1 in lymph node T follicular helper cells was evident for clozapine at all doses and time points evaluated (see Figure 35). In the case of CXCR5 expression, significant reductions were observed in mice dosed with clozapine from day 1 and evident in both lymph node (strongest signal for reduction) and spleen (see Figure 36). In addition, a reduction in expression of CCR7 was observed on germinal centre resident T follicular helper cells in both lymph node and spleen of mice treated with clozapine (see Figure 37).

Effect of Clozapine on T regulatory cells:

When used at the higher dose tested and from day 1 after immunisation, clozapine was seen to increase the proportion of CD4 ⁺ CD25 ⁺ Foxp3 ⁺ T regulatory cells (Tregs) in both lymph node and spleen (See Figure 40). In addition, clozapine when dosed from day 1 was seen to significantly upregulate the expression of CD25 on these cells (see Figure 41), but not alter Foxp3 expression itself (see Figure 42).

Conclusion

This study investigated the potential for clozapine to ameliorate CIA and its impact on major B cell subsets. The major findings of this study are as follows. a) Clozapine is extremely effective at delaying disease onset in the CIA model. b) Clozapine ameliorates the severity in CIA. c) Clozapine reduces the proportion of B220 ⁺ B cells in both spleen and lymph node. d) Clozapine reduces the proportion of splenic plasma cells. e) Clozapine results in substantial reduction in the proportion of lymph node follicular B cells (IgD Fas ⁺ GL7 ^hl ) in B220 ⁺ B cells and lowers their expression of GL-7. f) Clozapine demonstrated some ability to reduce pathogenic immunoglobulin, specifically anti collagen IgGl (at a dose of 10 mg/kg dosed from D15 after immunisation) in the context of the experimental conditions assessed (single time point immunoglobulin measurement). g) Clozapine markedly reduces the expression of PD1 and CXCR5, in addition to CCR7, on lymph node T follicular helper cells (PD1 ⁺ CXCR5 ⁺ ) without impacting upon the proportion of cells.

Taken together, these observations indicate that clozapine delayed disease onset, probably through multiple mechanisms likely to involve its impact on (secondary) lymphoid tissue and its ability to form functional germinal centres with subsequent impact on antibody producing B cells. Specifically, clozapine is seen to reduce germinal centre B cells in local lymph node [marked by expression of GL7 in immunised spleen/lymph node (Naito et al., 2007)] following immunisation. GL7 ^hl B cells exhibit higher specific and total immunoglobulin production in addition to higher antigen-presenting capacity (Cervenak et al., 2001). Thus the observation of a reduction in surface expression of the GL7 epitope with clozapine suggests an impact to lower functional activity of these B cells for producing antibody and presenting antigen.

In parallel, clozapine is seen to affect T follicular helper cells, a critical T cell subset which controls the formation of and coordinates the cellular reactions occurring within germinal centres that is essential for somatic hypermutation, isotype class switching and antibody affinity maturation, differentiating B cells into memory B cells or plasma cells. T follicular helper cells therefore specialise in promoting the T cell-dependent B cell response (Shi et al., 2018). In particular, while not affecting the overall proportion of T follicular helper cells, clozapine is seen to reduce PD1 (programmed cell death-1) expression which is essential for proper positioning of T follicular helper cells through promoting their concentration into the germinal centre from the follicle (Shi et al., 2018). PD1 is also required for optimal production of IL-21 by T follicular helper cells, with PD1-PD-L1 interactions (i.e. the cognate ligand of PD1) between T follicular helper cells and germinal centre B cells aiding the stringency of affinity-based selection.

Furthermore, clozapine was seen to reduce the expression of CXCR5 on T follicular helper cells. CXCR5 (CXC chemokine receptor 5) is regarded as the defining marker for these cells; upregulation of CXCR5 enables relocation to the T/B border and, through attraction to CXCL-13, the B cell zone of lymphoid tissue to allow T follicular helper cells to enter the B cell follicle (Chen et al., 2015).

Accordingly, reduced expression of CXCR5 on T follicular helper cells would impede their migration into B cell follicles and thereby reduce their ability to localise and interact with germinal centre B cells. Consistent with this, mice deficient in CXCR5 or selectively lacking CXCR5 on T cells display complete resistance to induction in CIA, in concert with reduced secondary lymphoid germinal centre formation and lower anti-collagen antibody production (Moschovakis et al., 2017).

Clozapine was also found to reduce expression of CCR7 on T follicular helper cells. CCR7

downregulation is regarded as an important mechanism through which activated CD4 ⁺ T cells overcome T zone chemokines which promote retention in the T zone (Haynes et al., 2007).

Importantly, promotion of normal germinal centre responses by T follicular helper cells requires a coordinate upregulation of CXCR5 and downregulation of CCR7 (Haynes et al., 2007). Thus, the balanced expression of CXCR5 and CCR7 is critical to fine tuning of T follicular helper cell positioning and efficient provision of B cell help (Hardtke et al., 2005). The observation that clozapine can influence both CXCR5 and CCR7 expression on T follicular helper cells is therefore consistent with an ability of clozapine to perturb positioning and proper function of these cells, vital for T cell support of production of high affinity antibodies in response to T dependent antigens.

Further highlighting the importance of germinal centre formation to the pathogenesis of CIA is the finding that syndecan-4 null mice, which exhibit lower numbers of B cells and deficient germinal centre formation in draining lymph nodes, are resistant to CIA (Endo et al., 2015). Given the critical importance of tight regulation of germinal centres to the maintenance of self-tolerance and prevention of pathogenic autoantibody production in autoimmunity, the impact of clozapine as demonstrated in the CIA model strongly supports its potential to mitigate pathogenic autoantibody production.

Example 5

Study of effect of clozapine and norclozapine on human plasma cell generation using an in vitro B cell differentiation system

An established in vitro platform (Cocco et al., 2012) was used to evaluate the impact of clozapine, its major metabolite norclozapine and a comparator antipsychotic drug, haloperidol, on the generation and differentiation and viability of human plasma cells.

Method

General:

The system employed is based on a published model (Cocco et al., 2012) which uses a CD40L/I L-2/1 L- 21 based stimulus to drive B-cell activation and differentiation in a 3-step process to generate plasmablasts and functional polyclonal mature plasma cells (See Figure 38). The final step of the culture (Day 6-9) was performed in the context of IFN-a driven survival signals and without stromal cells.

The experiment was performed using total peripheral blood B-cells isolated from healthy donors.

The experiment was performed from four independent donors.

Drug addition:

Compounds were sourced from Tocris and dissolved in DMSO at the following concentrations: Clozapine:

• 350ng/ml Clozapine (approximately equivalent to 500mg adult human dose)

• lOOng/ml Clozapine

• 25ng/ml Clozapine (approximately equivalent to 55mg adult human dose)

Norclozapine: • 200 ng/ml norclozapine

• 70 ng/ml norclozapine

• 15 ng/ml norclozapine

Haloperidol:

• 25 ng/ml Haloperidol

• 8 ng/ml Haloperidol

• 2 ng/ml Haloperidol

DMSO as diluent control at 0.1%. All DMSO concentrations were adjusted to 0.1% for all drug treated samples.

Drugs were added at two time points:

• day-3 of the culture (activated B-cell/pre-plasmablast), or

• day-6 of the culture (plasmablast)

Evaluation:

The cultures were evaluated 3 days after addition of the compound with day-3 drug additions evaluated at day-6 (plasmablast) and day-6 drug additions evaluated at day-9 (early plasma cell) (see Figure 38).

Evaluation encompassed:

Flow cytometric assessment of:

• phenotype (CD19, CD20, CD27, CD38, CD138)

• viability (7AAD)

• cell number (bead count)

Immunoglobulin secretion:

• ELISA analysis of total IgM/lgG from bulk supernatant collected at day 6 and day 9 of

respective cultures

Results

Cell phenotype:

Across all four donors the control DMSO samples demonstrated a transition to a plasmablast state from day 3 to day 6 with downregulation of CD20, upregulation of CD38 and variable upregulation of CD27 combined with retained CD19 expression and lack of CD138. On subsequent transfer into plasma cell maturation conditions the control cells showed progressive loss of CD20, downregulation of CD19 and upregulation of CD138 combined with further upregulation of CD38 and CD27 indicating transition to early plasma cell state. These findings indicate that the differentiation protocol worked in relation to phenotype and that all four samples were suitable as references for the in vitro differentiation system.

In terms of effects on phenotypic maturation none of the drugs at any concentration showed significant effects on the downregulation of the B cell phenotype as reflected in equivalent loss of CD20 and CD19 expression. None of the drugs at any concentration showed significant effects on the pattern of acquisition of C27 or CD138 expression at either day 6 or day 9 time points.

All three drugs showed a dose related effect on the expression of CD38 in one donor. This was modest at the day 6 time point but was significant at the day 9 time point with a substantial and reproducible shift in CD38 expression. However, this effect was not observed as a consistent effect across the other donors.

Cell number and viability:

Across all four donors the control DMSO samples demonstrated an expansion to the plasmablast state from day 3 to day 6 and contraction during the transition to plasma cell state. Based on an input activated B cell number at day 3 of 10 ⁵ the average expansion observed during the day 3 to day 6 culture was 12-fold. There was a 5-fold contraction that accompanied the maturation to the plasma cell state from 5x10 ^s input at day 6 to 10 ⁵ viable cells at day 9. It was concluded that the differentiation protocol worked in relation to cell number and that all four samples are suitable as references.

None of the drugs at any concentration impacted significantly on the number of viable cells at either day 6 or day 9. This was not affected whether considering total cell number or viable cell number per input cell. Based on equivalent input activated B cell number the degree of expansion from day 3 to day 6 was equivalent across all drugs and concentrations. Equally there was no effect on the viable cell number recovered at day 9 with any drug at any concentration.

Immunoglobulin secretion:

Across all four donors the control DMSO samples showed evidence of significant IgM and IgG secretion at across the day 3 to day 6 culture. This was continued into the day 6 to day 9 culture with predicted higher per cell estimated secretion rates in this second culture phase to the plasma cell stated. It was concluded that the differentiation protocol worked in relation to immunoglobulin secretion and that all four samples are suitable as references. In terms of immunoglobulin secretion there is greater variation between individual donors, but there were no clear trends in response to any of the three drugs at any dose. Normalising to DMSO as control provided the simplest view of the data and showed only minor shifts in the detected immunoglobulin in relation to IgG. Where changes are observed these follow inverse responses in relation to the dose for example norclozapine with one donor.

Conclusion

The results showed that none of the drugs are directly toxic to differentiating B-cells, nor do any of the drugs at any concentration show consistent effects on the ability of the resulting differentiated antibody secreting cells to secrete antibody.

In terms of phenotypic responses there is variability between the donors in relation to CD38 expression with one donor in particular showing an apparent dose dependent downmodulation in the window of differentiation between plasmablast (day 6) and early plasma cell (day 9). However this response did not reproduce as a consistent feature across the other donors tested.

Overall, therefore, the compounds as tested do not show a consistent inhibitory effect on the functional or phenotypic maturation of activated B-cells to the early plasma cell state and have no effect on viability of antibody secreting cells.

The in vitro system employed has limitations in terms of being a 'forced' B cell differentiation assay (as opposed to physiological expansion), with a focus on peripheral B cells, limited culture duration which may not reflect effects of very chronic exposure, and lack of the normal micro-environment of B cells in primary (e.g. bone marrow) or secondary lymphoid tissues, nor indirect regulation (e.g. through T follicular helper cells and/or IL-21). Notwithstanding these, the findings suggest that clozapine is unlikely to be acting directly on plasma cells or their precursors and that the

immunophenotypic findings in vivo reflect a more complex and/or indirect action. The findings from this in vitro study are consistent with the lack of reduction in overall B cell numbers (i.e. no evidence of generalized B cell depletion in patients taking clozapine).

Example 6

Healthy Human Volunteer Study - primary vaccination response to Typhim Vi

This study was an open-label, single-centre, non-randomised, controlled single-dose level study investigating the effects of short-term low-dose clozapine administration on B cell number and function in healthy volunteers following primary vaccination (i.e. antigenic challenge) with Typhim Vi. Background to vaccine selection - Typhim Vi

The vaccine used in this study was Typhim Vi which contains purified Vi capsular polysaccharide (Vi CPS) of Salmonella typhi (Ty2 strain). The Vi antigen, made of repeating units (l-4)-2-deoxy-2-N- acetyl galacturonic acid (Marshall et al., 2012), forms a polysaccharide capsule around S. Typhi and as a surface polysaccharide is regarded as a T-independent type 2 (T-l type II) antigen (MacLennan et al., 2014). Notably, Vi specific IgG responses to Typhim Vi vaccination have been used as a means of assessing the immune response to polysaccharide antigen in patients with suspected antibody deficiency (Bausch-Jurken et al., 2017; Evans et al., 2018). A key advantage of Typhim Vi in this regard (e.g. compared to Pneumovax) is the ease of interpretation of antibody responses given generally low background or pre-vaccination concentrations of Typhi Vi IgG.

T-dependent antigens refer to proteins which are processed and presented on M HC class II molecules to enable recognition by cognate CD4+ (helper) T cells. The response to immunisation with T cell dependent antigen involves the formation of germinal centres, transient structures that develop within peripheral lymphoid organs in which B cells proliferate, diversify their

immunoglobulin genes via somatic hypermutation (SHM) to generate high affinity antibodies, undergo class-switch recombination or differentiate into memory B cells or plasma cells (De Silva and Klein, 2015). Long-term use of clozapine in patients has been linked to a specific reduction in circulating class-switched memory B cells suggesting an impact on germinal centre function and/or formation (Ponsford et al., 2020).

In contrast, polysaccharides, as T cell independent antigens are traditionally regarded as stimulating short-lived B cell responses through cross-linking of the B cell receptor and promotion of differentiation to antibody secreting cells (plasma cells) with little production of memory B cells (Pollard et al., 2009). Splenic marginal zone (MZ) B cells are thought to be important in mediating the immune response to such T-independent antigens - notably infants, which lack a mature MZ B cell compartment, have poor responses to such vaccines. As a corollary, humans without a functional spleen are thought to be vulnerable to infections with encapsulated bacteria in part reflecting their inability to form protective MZ B cell derived antibody responses against T independent antigens (Bemark, 2015). Beyond these cells, in mice, Bib cells - regarded as innate immune cells localising to the peritoneal and pleural cavities and producing 'natural' IgM and IgA as a first line defence against encapsulated polysaccharide-expressing bacteria - also contribute to immunity induced by Vi antigen, although they are regarded as contentious in humans (Tangye, 2013). More recently, T cell-independent type II antigens have been shown to generate long-lived plasma cells via extra-follicular foci, as well as memory B cells, although the latter show low levels of SHM and class switching to secondary (i.e. non-lgM) isotypes (Obukhanych and Nussenzweig, 2006). T cell independent antigens are not thought to generate switched memory B cells (Rosado et al., 2013).

Accordingly the specific vaccine utilised enabled a specific evaluation of the potential for clozapine to impact upon T-l type II antigen responses, but not T-dependent or T-l type I antigens, the latter representing mitogenic stimuli that elicit polyclonal B cell activation via Toll-like receptors (e.g. LPS or CpG) (Obukhanych and Nussenzweig, 2006).

Method:

The study employed a parallel arm design (see Figure 39) recruiting two arms: an active arm

(receiving clozapine) and a time matched control arm, with both receiving Typhim Vi vaccination. The study design, employing an active arm with dosing initiated before vaccination during an uptitration phase, together with a control vaccine-only arm, enabled evaluation of: i) the normal humoral immune response to Typhim Vi vaccination (i.e. in controls); ii) any influence of clozapine on the primary vaccination response to the same antigenic stimulus; iii) the impact of short-term clozapine alone during the uptitration period immediately preceding vaccination.

Active Arm (Clozapine and Typhim Vi):

After a screening period (Day -35 to Day -8), eligible subjects were asked to return for the treatment period. Subjects in the active arm were then treated from Day -7 to Day 34.

This began as an in-patient treatment period (Day 7 to Day 3) of ~ 10 days in duration (up to 48 hours post-Typhim Vi dose on, i.e. Day 3). On Day -7 to Day -1, subjects were up-titrated from 12.5 mg to 100 mg clozapine over a 7-day period (finally reaching 100 mg as 25 mg a.m. and 75 mg p.m.). On Day 1 subjects received their morning dose of clozapine after which (approximately 1 h later) they were inoculated with Typhim Vi. On Day 3 subjects received the morning dose of clozapine (25 mg) prior to discharge and self-administered the evening dose (75 mg).

The out-patient treatment period was approximately 30 days in duration (from Day 4 to Day 34), with all out-patient clozapine doses self-administered. Subjects returned to the Clinical Unit on Days 7, 14, 21 and 28 for scheduled assessments. On Day 29 to Day 34, to minimise risk of cholinergic rebound symptoms, subjects down-titrated gradually from 100 mg to 12.5 mg clozapine and then stopped over a 6-day period (Day 29 to Day 34). Subjects returned to the Clinical Unit for final follow-up 63 days after inoculation of Typhim Vi.

Control arm (Typhim Vi only):

Screening assessments were carried out between Day -28 and Day -2. Eligible subjects were asked to return for the treatment period.

On Day 1 control subjects were inoculated with Typhim Vi and discharged on Day 2. Subjects then returned to the Clinical Unit on Days 7, 14, 21 and 28 and 63 for scheduled assessments.

Objectives

Primary Study Objective

The primary objective of this study was:

• To understand the effect of clozapine on primary vaccination response (change from baseline and fold-increase in specific anti-Typhim IgG at Day 28).

Secondary Study Objectives

The secondary objectives of the study included:

• To determine the effect of clozapine on circulating Ig levels.

• To determine the effect of clozapine on plasmablast levels.

• To provide general safety and tolerability information for clozapine in healthy subjects.

Exploratory Study Objectives

• To determine the effect of clozapine on class-switched memory B cells and other lymphocyte populations.

• To investigate any correlation between plasma concentration levels of clozapine with changes in B cell function.

Analysis sets

Study analysis sets included an immunogenicity set (all subjects who received at least 1 dose of clozapine or administration of Typhim Vi vaccine for whom at least 1 post-baseline immunogenicity measurement was taken) and a Per Protocol (PP) Set (all subjects in the immunogenicity set who had not violated any major entry criteria and did not deviate from the protocol. Subject disposition and demographics

Twenty-five (25) subjects were enrolled into the study (13 in the active arm [12 and 1 replacement] and 12 in the control arm). Twenty-three (23) subjects completed the study. One subject withdrew consent on Day 19 due to AE (somnolence [moderate, almost definitely related to IMP]) and was replaced, while another was withdrawn due to detection of isolated, asymptomatic mildly elevated cardiac troponin. One subject was withdrawn from treatment (not allowed to down-titrate) on Day 28 due to non-compliance with IMP administration (returned 33 tablets instead of 8), however the subject did remain in the study for safety considerations.

All subjects (100%) were Caucasian (84% male, 16% female) with mean overall age of 36.8 years. Subject demographics were similar in the active and control groups.

Results

Pharmacokinetic results

The geometric mean plasma concentration-time curves for clozapine are presented on a linear scale (see Figure 43). Plasma clozapine levels reached a maximum of 163 pg/L (geometric mean) at study Day 7, reflecting the end of residential observed dosing, but then gradually declining by >50% to 69 pg/L by study Day 28. Mean levels of plasma clozapine peaked at Day 7 post-vaccination and then reduced thereafter reaching their lowest levels at Day 28 suggesting possible variable treatment adherence beyond the residential up titration period in the active group. Individual plasma concentrations for clozapine following administration of clozapine (Denzapine; active group only) showed intersubject variability despite equivalent prescribed dosing (i.e. 100 mg daily clozapine).

Similarly, levels of norclozapine reached a peak at Day 14 post-vaccination, consistent with its longer half-life (approximately double that of parent clozapine) (see Figure 44). Individual plasma concentrations for norclozapine following administration of clozapine (Denzapine; active group only) also showed intersubject variability.

Pharmacodynamic results - Typhi Vi IgG

In most subjects in both groups, IgG specific for salmonella Typhi Vi was undetectable at screening.

IgG specific for salmonella Typhi Vi levels increased in both active and control arms following vaccination with Typhim Vi. Both groups displayed robust increases from baseline in Day 28 IgG (specific for salmonella Typhi Vi) concentration data in absolute terms, but with marked inter- individual differences in the magnitude of primary immune response to Typhim Vi inferred from specific IgG measurement (see Figure 45). There was no statistically significant difference in the Day 28 antibody levels between the active and control groups (P = 0.1932) or when expressed as fold increase.

Table: Statistical Analysis of Salmonella Typhi Vi IgG Antibody Levels (U/mL) at Day 28

(Immunogenicity Set)

Results obtained using an ANCOVA with treatment group as a fixed effect and baseline (screening) as a covariate.

ANCOVA = analysis of covariance, i.m = intramuscular

Beyond magnitude and fold-change from baseline, this study was not designed to address or able to exclude whether clozapine had differential impact on antibody avidity (to Vi antigen), or specific impact on specific IgG subclasses to Vi and/or on other specific immunoglobulin isotypes (including IgM and/or IgA to Vi).

PD results - total circulating immunoglobulins

Total IgA

Vaccination had no clear impact on circulating total IgA in the control group. There was some tendency to lower total IgA versus baseline in the active group by Day 28, but this did not achieve statistical significance.

Total IgG

A reduction in total IgG (~0.6 g/L) was observed with low-dose clozapine from during the uptitration period pre-vaccination and reaching a nadir at day 7 which approached statistical significance (P=0.0863). This trend was not apparent at later time points suggesting a potential confounding effect of the vaccination and/or transient impact on total circulating IgG by clozapine (see Figure 46). Similarly, analysis of fold-increase total IgG data between active and control groups revealed a lower fold change in circulating IgG at day 7 post-vaccination which approached statistical significance (P=0.06).

Total IgM

Clozapine was associated with a trend (P=0.18) to progressive reduction in total IgM below baseline throughout the period of active dosing, maximal at Day 28 (see Figure 47 A). There was evidence of a partial recovery toward baseline in the post-dosing phase (i.e. by Day 63), very little impact of vaccination on total IgM in the control group and greater variance in IgM levels in the clozapine group compared to the control group at all time points post-baseline. Notably, IgM has the shortest serum half-life of all the immunoglobulins measured (IgM, 5 days; IgA, 6 days; IgG, 23 days).

Overall, a similar pattern of results was observed in the PP analysis set (see Figure 47B) with a significant reduction in IgM levels at Day 63 (LSMean 0.84 vs 0.97 g/L, P=0.0197) and significantly greater IgM fold change (LSMean fold-increase 0.87 vs 0.99, P=0.0496) in active vs control groups, consistent with an effect of clozapine to reduce total IgM.

Specific IgG to pneumococcus and to tetanus

Closely mirroring the total IgG data, a significant reduction in fold-change specific IgG to

pneumococcus was observed on Day 7 in the active vs control groups (P=0.0308). After Day 7, there was no statistical difference between active and control groups suggesting either a transient effect of clozapine and/or superimposed impact of the primary vaccination.

The pattern of response of specific anti-pneumococcal IgG in the active group - with reduction commencing with dosing pre-vaccination and continuing to a nadir at Day 7 followed by partial recovery to baseline - closely mirrored that of total IgG, consistent with a biological effect.

There was no clear evidence of impact of short-term low-dose clozapine dosing on specific IgG to tetanus.

PD - Plasmablast levels (defined as CD19+ CD27+ CD38+ IgM- IgD- cells)

Circulating plasmablasts were present at very low levels at baseline and remained so throughout the study in the control group. Similarly, plasmablasts were present at a very low level in the active group with a tendency to reduction pre-vaccination (i.e. from Day -7 to Day 1). Primary vaccination with Typhim Vi did not induce a convincing or robust plasmablast response in the control group (with plasmablasts defined as CD19+ CD27+ CD38+ IgM- IgD- cells). The reasons for this are unclear but may relate to the vaccine used and/or the labelling strategy used to define plasmablasts.

While the Day 7 (post-vaccination) plasmablast response was not significantly different between the clozapine treated (active) and control groups, the lack of any induction of plasmablasts in the control group means that the study was unable to confirm or exclude an ability of clozapine to impact on a vaccine-induced plasmablast response. While no clear impact of vaccine on total plasmablasts was discerned, Vi-specific cellular humoral immune response, specifically Vi-specific antibody-secreting cells were not formally evaluated. Exploratory PD analyses

Autogated flow cytometry - white cell blood count & major lymphocyte populations

White cell count

In the active group, there was an increase in total white blood cell count (i.e. a leucocytosis) apparent from baseline (i.e. commencing pre-vaccination in response to clozapine) during clozapine uptitration, which was statistically significant and reaching a peak by Day 7 (P=0.0011) and remained significantly elevated to Day 63 (P=0.0075), i.e. final timepoint of study evaluation off clozapine, in comparison to control subjects.

Table: Statistical Analysis of Change from Baseline White Blood Cell Count (xlO ^A 9/L)

(Immunogenicity Set)

Results obtained using an ANCOVA on change from baseline results with treatment group as a fixed effect and baseline (Day -7 pre-IMP for the active group and Day 1 pre-NIMP for the control group) as a covariate.

ANCOVA = analysis of covariance, i.m = intramuscular, IMP = investigational medicinal product, NIMP = non- investigational medicinal product

There was a moderate positive (r = 0.474) and highly statistically significant (P=0.0006) correlation between white blood cell count and plasma clozapine levels, consistent with a linear relationship between drug concentration and immune parameter at alpha=0.05. Total lymphocytes

A small increase in total lymphocyte count (i.e. a component of the white blood cell count) was apparent in the active arm from Day -7 to Day 1 followed by a trend to reduction at Day 7.

Lymphocyte count tended to rise progressively further in the active group from around Day 7, with change from baseline levels (i.e. Day -7) reaching statistical significance on Day 14 (P=0.0440) and Day 28 (P=0.0001) when compared to control. In contrast there was very little overall change in total lymphocyte count over time in the control group.

Table: Statistical Analysis of Change from Baseline Lymphocyte Count (xlO ^A 9/L) (Immunogenicity Set)

Results obtained using an ANCOVA on change from baseline results with treatment group as a fixed effect and baseline (Day -7 pre-IMP for the active group and Day 1 pre-NIMP for the control group) as a covariate.

ANCOVA = analysis of covariance, i.m = intramuscular, IMP = investigational medicinal product, NIMP = non- investigational medicinal product Clozapine use in the active group was therefore associated with a significant and marked

lymphocytosis commencing from dosing (i.e. pre-vaccination), with a transient reduction at Day 7 post-vaccination only (suggesting a short-term influence of the latter). There was no correlation between plasma clozapine levels and change in lymphocyte count from baseline.

Total (CD19+) B cells Overall, there was no significant effect of short-term low dose clozapine administration on circulating levels of total B cells from either the auto-gated or manually gated flow cytometry data during the course of the study. However, there was a modest negative (r = -0.307) and significant (P=0.0321) correlation between autogated CD19+ B cells and plasma clozapine levels (see Figure 48), suggesting a linear relationship between concentration and parameter at alpha=0.05. Total (CD3+) T cells

There was no impact of Typhim Vi vaccination on total T cell count in the control group.

CD3+ (T lymphocyte) count increased from baseline to D1 (i.e. pre-vaccination/effect of clozapine alone) in the active arm and then tended to progressively rise again in the active group from around Day 7, with change from baseline levels reaching statistical significance on Day 14 (P=0.0311), Day 28 (P=0.0008) and Day 63 (P=0.0231) when compared to control. The pattern of change in the active arm, i.e. immediate rise post-vaccination followed by transient reduction in the 7 days following primary vaccination and then a progressive marked increase, closely mirrored that of the total lymphocyte count, reflecting the fact that T cells are the major cellular component of the total lymphocyte count (laboratory reference range: 57.2-86.8%). Overall, these finding suggest clozapine stimulated an increase in the total CD3+ T cell count.

Table: Statistical Analysis of Change from Baseline CD3 (T Lymphocyte) Count (xlO ^A 9/L) (Immunogenicity Set)

Results obtained using an ANCOVA on change from baseline results with treatment group as a fixed effect and baseline (Day -7 pre-IMP for the active group and Day 1 pre-NIMP for the control group) as a covariate.

ANCOVA = analysis of covariance, i.m = intramuscular, IMP = investigational medicinal product, NIMP = non- investigational medicinal product

CD4+ (helper) T cells CD4+ (helper T lymphocyte) count tended to increase from baseline to Day 1 in the active group

(reflecting the effect of clozapine alone pre-vaccination) and then tended to rise further in the active group from around Day 7, with change from baseline levels reaching statistical significance on Day 14 (P=0.0032), Day 28 (P=0.0005) and Day 63 (P=0.0138) when compared to control. These findings indicate an increase in the CD4+ subset of T cells induced by clozapine as a contributor to these larger lymphocyte compartments. In contrast, primary vaccination with Typhim Vi had minimal or no effect on the levels of CD4+ T cells in the control group.

This observation was consistent whether determined by autogated or from manually gated multiparameter flow cytometry data. There was no correlation between plasma clozapine levels and change in CD4+ T cell count from baseline. CD8+ (cytotoxic) T cells

There was no impact of Typhim Vi vaccination on CD8+ T cell count in the control group.

Short-term clozapine use alone (i.e. pre-vaccination from Day -7 to Day 1) did not influence circulating CD8+ T cell count.

However, following vaccination there was a modest increase in total CD8+ T cells over baseline in the active group which achieved significance versus control at a variable single timepoint based on either the autogated (Day 28, P=0.0254) or manually gated flow cytometry data. Both the magnitude of this increase and number of timepoints at which this achieved statistical significance was markedly lower than that observed for the CD4+ T cell compartment. A weak positive (r=0.308) and significant (P=0.0313) correlation was found between plasma clozapine level and change in circulating CD8+ T cell count from baseline (using the manually gated dataset).

NK cells

Natural killer (NK) cell count tended to reduce slightly to D1 (reflecting the impact of clozapine alone pre-vaccination) and then progressively rise in the active group from around Day 1, with change from baseline levels reaching statistical significance on Day 28 (P=0.0059) and Day 63 (P=0.0128) when compared to control. In contrast, there was very little change in the levels of NK cells in the control group, consistent with no impact of Typhim Vi vaccination on NK cell count in the control group. There was no correlation between plasma clozapine levels and change in NK cell count from baseline (r=0.147).

Manually gated multiparameter flow cytometry - B cell panel

CD21- CD38lo B Cells

There was an apparent reduction in CD21- CD38lo B cells from baseline to D1 in the active group, suggesting an acute influence of clozapine alone pre-vaccination. Overall, there was no statistically significant difference in change from baseline CD21- CD38lo B cell count between active and control groups. There was a weak negative (r = -0.345) and significant (P=0.0153) correlation between CD21- CD38lo B cell count and plasma clozapine levels, suggesting a linear relationship between concentration and parameter at alpha=0.05. A similar pattern of results was observed in the PP analysis set.

Class Non-Switched Memory B Cells (CD19+ CD27+ CD38lo lgM+)

There was a trend to reduction in class non-switched memory B cells from Day -7 to Day 1, suggesting an acute impact of clozapine on this B cell subset pre-vaccination, followed by a rise to Day 7 and no further change. There was no statistical difference in change from baseline class non- switched memory B cell count between active and control groups.

Class Switched Memory B Cells (CD19+ CD27+ CD38lo IgM- IgD-)

Class-switched memory B cells tended to fall from baseline to D1 in the active group (i.e. in response to clozapine alone). There was no statistical difference in change from baseline class switched memory B cell count between active and control groups.

There was a statistically significant but small degree of correlation between class switched memory B cell count and plasma clozapine levels (r=-0.284). lgD+ CD27+ B Cells lgD+ CD27+ B cell count tended to fall in the active group between Day -7 and Day 7, suggesting an early effect of clozapine to reduce circulating levels of this B cell subset, with a statistically significant difference in change from baseline observed between active and control groups on Day 7

(P=0.0161), before counts started to rise. There were minimal changes from baseline observed in the control group over the course of the study. There was a moderate negative (r = -0.443) and statistically significant (P=0.0014) correlation between lgD+ CD27+ B cell count and plasma clozapine levels (see Figure 49), suggesting a linear relationship between concentration and parameter at alpha=0.05.

IgM hi IgD lo Memory B Cells (CD19+ CD27+ CD38lo lgM+ IgD-)

There was a marked reduction in IgM hi IgD lo memory B cells in the active arm from Day -7 to Day 1, remaining below baseline for the duration of dosing with recovery by Day 63. The control group showed some reduction 28 days after vaccination of uncertain significance.

Table: Statistical Analysis of Change from Baseline IgM hi IgD lo Memory B Cell Count (xlO ^A 9/L) (Immunogenicity Set)

Results obtained using an ANCOVA on change from baseline results with treatment group as a fixed effect and baseline (Day -7 pre-IMP for the active group and Day 1 pre-NIMP for the control group) as a covariate.

ANCOVA = analysis of covariance, i.m = intramuscular, IMP = investigational medicinal product, NIMP = non- investigational medicinal product

Evaluation of individual level data for this memory B cell subset expressed as either absolute concentration or as % total B cells (%B) (see Figure 50A and B) showed a marked reduction in levels in the active group occurring at the first time point measured after dosing (i.e. from Day -7 to Day 1), with evaluation of individual level concentration data consistent with a rapid effect of clozapine to eliminate this subset from the circulating compartment in days starting before vaccination and which remained near undetectable for the duration of dosing in the active group.

In common with several other memory B cell subsets, there was a moderate negative (r = -0.417) and significant (P=0.0029) correlation between IgM hi IgD lo memory B cell count and plasma clozapine levels, suggesting a linear relationship between concentration and parameter at alpha=0.05. The apparent association with reduction in this subset appeared most consistent at plasma clozapine levels above the low 200's (pg/L). A similar pattern of results was observed in the PP analysis set.

MZ-Like (marginal zone-like) B Cells (CD19+ CD27+ CD38+ lgM+ lgD+)

In the active group the MZ-like B cell count tended to fall following initiation of clozapine (Day -7) to reach a nadir at Day 1 followed by a return to baseline 2 weeks after vaccination. There was no statistical difference in change from baseline MZ-like B cell count between active and control groups. There was no correlation between MZ-like B cell count and plasma clozapine levels.

Naive B Cells (CD19+ CD27- lgM+ lgD+)

This B cell subtype tended to fall in the active group after initiating clozapine reaching a nadir on the day of vaccination (Day 1) followed by a return to baseline by approximately Day 7. The control group showed a reduction in naive B cell count immediately following vaccination to Day 7 tending to then rise to Day 14. There was no statistical difference in change from baseline naive B cell count between active and control groups. There was no correlation between naive B cell count and plasma clozapine levels (r= -0.179). Transitional B Cells (CD19+ CD27- CD24++ CD38++ lgM+ lgD+)

Transitional B cell counts tended to fall immediately in the active group (pre-vaccination) reaching a nadir at Day 1 followed by a progressive rise, with change from baseline levels reaching statistical significance on Day 28 (P=0.0284) when compared to control. In contrast, the transitional cell count tended to fall in the control arm to Day 7 and peaked at Day 14, suggesting a dynamic effect of vaccination alone on this cell subset. There was a trend to a weak negative correlation between transitional B cell count and plasma clozapine levels (r= -0.221).

Summary of results for B cell subsets

There was minimal or no impact of Typhim Vi vaccination on several B cell subsets in the control group, including: total (CD19+) B cells, CD21- CD38lo and plasmablasts.

Typhim Vi vaccination had a complex impact on the abundance of several circulating B cell subsets in the control group with a signal for a biphasic response apparent consisting of an initial fall in circulating levels shortly after vaccination (typically Day 7), suggestive of a potential initial sequestration/homing of these cells in peripheral lymphoid tissue and/or reduced generation, followed by a subsequent peak (typically at Day 14) post-vaccination, consistent with increased generation and/or release into the circulation.

This pattern was evident for: class non-switched memory, class-switched memory (although nadir at Day 14 with return to baseline rather than peak), MZ-like, naive, and transitional B cells. Notably the nadir of class non-switched memory and switched memory was separated by a week in the control group with a peak in the former by Day 14 suggesting an increased generation of class non-switched memory B cells in response to primary Typhim Vi vaccination. These responses were absent in the active group suggesting an attenuation of this process by clozapine.

In general this pattern was less evident or absent for the active group suggesting complex modulation of the B cell response to Typhim Vi vaccination by clozapine, including: an absence of apparent nadir in circulating class non-switched memory (seen at Day 7 in control) and class- switched memory B cells (seen at Day 14 in control); absence of clear nadir and peak at Days 7 and 14 in MZ-like, transitional and naive B cells.

Initiation of clozapine alone (inferred as changes from Day -7 to Day 1, i.e. pre-vaccination) was associated with a rapid reduction below baseline mean circulating values in virtually all B cell subsets evaluated, spanning the breadth of B cell development interrogated, including: CD21- CD38 lo, class non-switched memory, class-switched memory (tendency), lgD+ CD27+ (tendency), IgM hi IgD lo memory, MZ-like, naive B, plasmablasts and transitional B cells.

Specifically, clozapine use was associated with an almost complete disappearance of the circulating IgM hi IgD lo memory B cell subset (defined as CD19+ CD27+ CD38lo lgM+ IgD-) within 7 days of initiating dosing.

Clozapine's apparent suppressive effect on the circulating levels of this memory B cell subset were maintained during the period of dosing with full recovery to baseline in the post-dosing phase (to Day 63).

There were significant consistently negative correlations (ranging from weak to moderate) between plasma clozapine levels and multiple B cell subsets, particularly memory B cell subsets suggestive of a potential dose-response association, including: total CD19+ (i.e. total B cell count), CD21- CD38lo, class-switched memory, lgD+ CD27+, and IgM hi IgD lo memory B cells.

Manually gated multiparameter flow cytometry - T cell panel

CD4+ T Cells (helper T lymphocytes)

CD3+ CD4+ T cell count tended to rise in the active group from the start of dosing (between Day -7 and Day 1) with a slight reduction in Day 7 to then continue to increase and from Day 7) onward, with change from baseline levels versus control reaching statistical significance on Day 14

(P=0.0013), Day 28 (P=0.0004) and Day 63 (P=0.0264). The control group showed a mild reduction in CD4+ T cell count at Day 7 in response to vaccination, with minimal change thereafter. This overall pattern of change for both groups very closely mirrored that obtained by autogated measurement of CD4+ T cells.

There was no significant correlation between CD3+ CD4+ T cell count and plasma clozapine levels.

CD8+ T Cells (cytotoxic T lymphocytes)

Initiation of clozapine alone had no immediate apparent impact on circulating CD8+ T cell count (i.e. from Day -7 to Day 1) in the active group. In both groups, CD8+ T cell count tended to fall slightly immediately after vaccination (Day 7), this then remained relatively stable in the control group. However, CD3+CD8+ T cell count tended to rise in the active group from Day 7, with change from baseline levels reaching statistical significance on Day 28 (P=0.0246) i.e. the final day of full dosing for the active group, but not Day 63 (P=0.2579) when off clozapine in the active group .

This overall pattern of change for both groups very closely mirrored that obtained by autogated measurement of CD8+ T cells. There was a weak positive (r = 0.308) and significant (P=0.0313) correlation between CD8+ T cells and plasma clozapine levels.

CD4+ Central Memory Cells (CD3+ CD4+ CD27+ CD197+ CD45RA-)

While there was no statistical difference in change from baseline CD4+ central memory cell count between active and control groups, the overall pattern of change in this subset over time was similar to that observed in the total CD4+ T cell count, i.e. a tendency to increase in the active arm upon dosing (Day -7 to Day 1), followed by a slight reduction in both groups at Day 7 post-vaccine followed by either a maintenance of increased levels compared to baseline in the active group or a gradual return to baseline over the course of the study in the control group.

There was a moderate positive (r = 0.402) and significant (P=0.0042) correlation between CD4+ central memory cells and plasma clozapine levels, suggesting a linear relationship between concentration and parameter at alpha=0.05.

CD4+ Naive T Cells (CD3+ CD4+ CD27+ CD197+ CD45RA+)

CD4+ naive T cell count tended to rise on initiation of clozapine in the active group with only a slight reduction at Day 7 in the context of recent vaccination, to then further increase. In contrast, CD4+ na ^' ive T cells tended to fall following vaccination in the control group by Day 7 and remain lower than baseline during the study. Specifically, CD4+ naive T cell count tended to be higher in the active group (rising from Day 7), with change from baseline levels reaching statistical significance on Day 7 (P=0.0202), Day 14 (P=<0.0001), Day 28 (P=0.0002) and remaining significant at Day 63 (P=0.0153)

There was no correlation between CD4+ naive T cell count and plasma clozapine levels (r= 0.063).

CD8+ Central Memory T Cells (CD3+ CD8+ CD27+ CD197+ CD45RA-)

CD8+ central memory T cells tended to fall in response to vaccination in the control group, starting immediately from Day 1 to Day 7, and remaining lower than baseline for the duration of the study. CD8+ central memory cell count tended to be higher in the active group with a tendency to rise from initiation of dosing but only achieving significance when rising from Day 28), with change from baseline levels reaching statistical significance on Day 63 (P=0.0147).

There was a weak positive (r = 0.354) and significant (P=0.0125) correlation between CD8+ central memory T cells and plasma clozapine levels, suggesting a linear relationship between concentration and parameter at alpha=0.05. CD8+ Naive T Cells (CD3+ CD8+ CD27+ CD197+ CD45RA+)

Initiation of clozapine alone had no immediate apparent impact on circulating CD8+ naive T cell count (i.e. from Day -7 to Day 1) in the active group. In both groups, CD8+ naive T cell count tended to fall immediately after vaccination (nadir at Day 7), this then remained relatively stable in the control group at a level below baseline. In contrast, CD8+ naive T cell count tended to rise in the active group from Day 7, with change from baseline levels reaching statistical significance when peaking on Day 14 (P=0.0068) and remaining significant at Day 28 (0.0018), then fell back towards baseline thereafter.

Table: Statistical Analysis of Change from Baseline CD8+ Naive T Cell Count (xlO ^A 9/L)

(Immunogenicity Set)

Results obtained using an ANCOVA on change from baseline results with treatment group as a fixed effect and baseline (Day -7 pre-IMP for the active group and Day 1 pre-NIMP for the control group) as a covariate.

ANCOVA = analysis of covariance, i.m = intramuscular, IMP = investigational medicinal product, NIMP = non- investigational medicinal product

The overall pattern of change broadly mirrored that of total CD8+ T cell count in both groups. There was no correlation between CD8+ naive T cells and plasma clozapine levels.

Summary of results for T cell subsets

In contrast to the complex influence of Typhim Vi vaccination on the abundance of circulating B cell subsets in the control group, vaccination generally resulted in a uniform modest depression in circulating ! cell counts.

Specifically, in the control group there was a trend to a slight monophasic reduction following Typhim Vi vaccination, with a nadir generally maximal at Day 7 post-vaccination, including the following T cell subsets assessed: CD4+ T cells, CD8+ T cells, CD4+ central memory T cells, CD4+ naive T cells, CD8+ central memory T cells (nadir at Day 28), CD8+ naive T cells. The magnitude of any change in these subsets was generally significantly less than that in the active group.

Typhim Vi vaccination induced a transient mild increase in PD1+ helper T cells (CD3+ CD4+ CD197lo CD279+ CD45RA-) and PD1- helper T cells (CD3+ CD4+ CD197lo CD279- CD45RA-) at Day 7 post vaccination in the control group with a return to baseline by Day 14 and minimal further change. These cells are likely to be non-naive CD4 T cells (i.e. lack CD45R and have low/absent expression of the homing receptor CD197/CCR7).

Initiation of clozapine alone (inferred from changes from Day -7 to Day 1, i.e. pre-vaccination) was associated with an increase or trend to above baseline mean circulating values in almost all CD4+ T cell subsets evaluated, including: total CD4+, CD4+ central memory, CD4+ naive, and CD197lo CD279- CD45RA- ('PD1- T helper') T cells.

Following vaccination, in general the increase in total CD4+ T cells tended to continue except for a transient decline at Day 7, suggesting that clozapine may act to stimulate CD4+ T cell proliferation with potential transient sequestration of such cells shortly after vaccination (at Day 7). This induction generally peaked at Day 14 and was frequently maintained above baseline at the final timepoint of assessment (Day 63), consistent with a prolonged influence of clozapine on CD4+ T cells that persisted after dosing (and plasma drug exposure) had ceased.

In contrast, initiation of clozapine alone (inferred from changes from Day -7 to Day 1, i.e. pre vaccination) led to no changes in total CD8+ T cells (whether from autogated or manually gated data) and minimal or no effect alone on CD8+ central memory and CD8+ naive T cell numbers.

Following vaccination, in the active group there was a tendency for a transient reduction in total and naive CD8+ T cells at Day 7 followed by an increase to peak at Day 14, with either a persistent mild elevation (total CD8+) or return to baseline (naive CD8+ T cells) by Day 63. The latter increases were markedly lower than those observed in total CD4+ and naive CD4+ T cells in the active group.

Conclusion

Primary vaccination response to Typhim Vi

Vaccination induced robust increases in both groups although with marked inter-individual variation as has been described in healthy controls (see Figure 45) (Evans et al., 2018). With the dosing paradigm and sample size employed, there was no effect of clozapine identified on the primary vaccination response to Typhim Vi measured as specific anti-Typhi Vi IgG, with no significant difference observed between active and control groups in the Day 28 IgG antibody specific for Typhi Vi levels and fold-increase data.

Notably, the vaccine-elicited antibody response to Vi polysaccharide is dominated by Vi IgA and Vi lgG2, with the former showing the highest fold-change (Dahora et al., 2019). While an influence of clozapine on these specific isotypes is not excluded by the present study, this finding suggests that clozapine administered at low dose (100 mg) and commenced a week prior to vaccination has no significant impact on T-l type II antigen-mediated extrafollicular antibody responses.

The nature of the vaccine employed, based on Vi CPS, means that a specific effect of clozapine on primary immune response to T-D, including germinal centre formation, or T-l type I antigens is unaddressed. Specifically, recent studies have highlighted that Vi CPS vaccination does not result in induction of detectable circulating T follicular helper cells or Vi-specific IgG memory B cells, consistent with lack of a germinal centre response upon immunisation with Typhim Vi (Jin, 2018).

Development of specific typhi Vi IgG reflects a single end-point response to Typhim Vi vaccination. Notwithstanding the lack of impact on specific Vi IgG production, clozapine use was associated with significant differences in the kinetics of response of multiple adaptive immune cells following vaccination compared to controls, particularly in B cell subsets, suggesting subtle effects on the primary vaccination response not captured solely by a focus on specific IgG alone. Whether clozapine exerted a differential impact on specific anti-Typhi Vi IgM and/or IgA is unknown.

Impact on circulating immunoglobulins and other specific IgG

Total levels of IgM (see Figure 47) and IgA showed greater variance in the active group than the control group at all timepoints, suggesting a potential variable influence of clozapine on

immunoglobulin levels. Human immunoglobulins vary in their serum half-life of human

immunoglobulins, ranging from 5 days for IgM, 6 days for IgA and 23 days for IgG (Lobo et al., 2004), accordingly an impact of clozapine on immunoglobulin production in the context of short-term dosing would be expected to be most apparent for IgM and/or IgA and toward the end of the full dosing period (i.e. D28). In this regard, in the immunogenicity set a non-significant tendency to reduction in IgA from Day 14 to Day 28 and a progressive fall in IgM maximal by Day 28 with partial recovery by Day 63 (see Figure 47A) both in the active arm suggest a potential impact of clozapine on these immunoglobulins. Substantiating an impact of clozapine to progressively lower total IgM, analysis of the per protocol dataset showed a significant reduction in IgM levels and fold-change from baseline in the active versus control groups (see Figure 47B). While exhibiting the longest serum half-life of al immunoglobulins, total IgG decreased in both groups, more so in the active group starting pre-vaccination and maximal by Day 7 post-vaccine (see Figure 46). Intriguingly this pattern of transient reduction was closely mirrored for IgG specific for pneumococcus in the active arm, corresponding to a ~7 mg/L mean reduction below baseline (with median levels of anti-pneumococcal IgG reported as 40 mg/L in one study) (Parker et al., 2016), suggesting that this finding was unlikely to be purely due to chance.

The mechanisms underlying the long-term maintenance of anti-pneumococcal antibody levels are unclear, but may involve production by long-lived plasma cells regarded as the/a major source of serum immunoglobulins (Benner et al., 1981), bystander or polyclonal activation of B cells and/or re stimulation of memory B cells, e.g. by antigen persisting on follicular dendritic cells and/or nasopharyngeal exposure to pneumococci (Clutterbuck et al., 2006). The significant lowering of specific IgG to pneumococcus in the active group may reflect the disruption by clozapine of one of these processes, although given the long half-life of IgG and the presumed stability of the long-lived plasma cell compartment in the absence of a significant contribution from memory B cells (Ahuja et al., 2008), the biological significance of this observation is challenging to explain. Notably there was a tendency to reduction in total immunoglobulins even in the control group, although to a much lesser extent, raising the possibility that a factor associated with Typhim Vi vaccination per se played a role in the observed reductions in IgG, analogous to the reciprocal relationship described for splenic short-lived antibody-producing cell expansion and reduced long-lived plasma cell numbers in Staphylococcus aureus infection (Keener et al., 2017).

Lack of discernible induction of circulating plasmablasts

There was no discernible significant induction in total circulating plasmablasts after vaccination in either group. Accordingly, the Day 7 (post-vaccination) plasmablast response was not significantly different between the active and control groups. Vi-specific IgG antibody secreting cells have been detected post-Vi CPS vaccination using highly sensitive ex vivo enzyme-linked immunosorbent spot (ELISpot) assay, typically peaking at Day 7, although in a significant proportion of patients there appears to be no clear peak (Jin, 2018). In this study, (non-antigen-specific) plasmablasts were quantitated in peripheral blood by flow cytometry defined using the surface markers: CD19+ CD27+ CD38+ IgM- IgD-. While there was some tendency to reduction in plasmablasts in the active group following clozapine initiation, this was not sustained following vaccination. In the absence of a significant induction of plasmablasts in the control group, the study is unable to determine clozapine's ability to influence plasmablast induction.

The reason for lack of apparent induction of plasmablasts by the vaccine may relate to several factors, including: nature of the vaccine employed and potential limited ability to infer a signal from a bulk plasmablast pool using flow cytometry (rather than measurement of Vi specific plasmablasts by ELISpot), surface marker labelling strategy (including exclusion of IgM plasmablasts which may have contributed significantly to the overall signal).

Variability in PK of clozapine following repeat dosing

The licenced dose of clozapine in the setting of treatment resistant schizophrenia is up to 900 mg per day with a usual dose stipulated at 200-450 mg per day. This study evaluated the immunological effects of repeated low-dose clozapine (100 mg daily), with PK data indicating intersubject variability in plasma concentrations of clozapine and its major metabolite (norclozapine), reaching geometric mean levels of ~163 ug/L and 103 ug/L on Day 7 and 14 respectively (see Figures 43 and 44), both maximal for each analyte.

Therapeutic use of clozapine is known to lead to highly variable drug concentrations between individual patients adhering to the same dosing regimen. Factors contributing to the intersubject variability of clozapine include age, sex, smoking status (latter not relevant in the current study), CYP1A2 activity (a determinant of clozapine elimination), ethnicity and specific genetic variants (Jovanovic et al., 2020).

A threshold plasma concentration for clozapine of between 350-420 pg/L reported to be associated with greater likelihood of clinical response in the context of schizophrenia and related psychotic disorders. Such a threshold has not been established flor clozapine's immune actions but, overall, the data in this study are consistent with a low dose paradigm and indicative of potentially clinically relevant immune effects at these levels. Evidence to support a potential plasma exposure-response effect with a series of B cell subsets, in particular, suggest that higher doses are likely to have yielded greater signals for changes in immune parameters. With a mean elimination half-life of 9-17 hours, steady-state levels of clozapine are expected after "7-10 days of dosing (Jovanovic et al., 2020). Consistent with this, peak plasma levels were identified at Day 7 of dosing but then tended to fall thereafter (see Figure 43). These findings suggest that the plasma levels of clozapine at the time of vaccination and in the immediate post -vaccination period, representing the early stages of the B cell response, was likely to have been rising, variable between subjects and not fully reflective of the immune potential of a 100 mg dose (i.e. having achieved at least steady state at the time of vaccination). The progressive reduction in levels of clozapine and norclozapine (see Figure 44) after their peak at Day 7 or Day 14 suggest variable adherence to dosing in the community and represent a further factor to take into consideration when interpreting the apparent variable influence of clozapine on specific immune cell subsets over the short course of the study. A further consideration is that attainment of steady state in terms of drug exposure is not necessarily the same as having reached steady state for PD effect. These factors are likely to have contributed to the significant variance in the data obtained in the active group and reduce the study's ability to identify statistically significant influences of clozapine, compounded by the small sample size and known variability in response to the immune stimulus used.

Exploratory PD findings

Clozapine induces a leucocytosis, lymphocytosis and, in the context of Typhim Vi vaccination, an increase in circulating NK cells

Clozapine use was associated with a clear induction of leucocytosis, in turn substantially reflecting a neutrophilia and lymphocytosis upon dosing with clozapine. In the case of both leucocytosis and lymphocytosis, these commenced in the pre-vaccination stage and remained present in the post dosing period at Day 63, suggesting a sustained influence of clozapine to induce increased generation of these cells that persisted even in the absence of drug exposure. Whether these reflect a re-programming of progenitor cells by the drug and/or a residual initial, non-specific proliferative effect of clozapine on these immune cell types is unclear. Notably, other than leucocytosis, there was no clear evidence to support a (linear) dose-response relationship of clozapine on induction of these cell subtypes in the context of the dose range explored in the study (up to 100 mg).

The lymphocytosis observed in response to clozapine was driven substantially by an increase in circulating T cells, in particular CD4+ T cells, commencing before vaccination suggesting a direct/indirect effect to increase peripheral levels of this T cell subset in particular. Whether this reflects a direct/indirect (e.g. through release of cytokines) proliferative effect of clozapine on CD4+ T cells and/or an impact on homing in secondary lymphoid tissue (i.e. reduced sequestration via effects on chemokine expression) is unknown.

A further marked induction of NK cells was also observed in the active arm in response to vaccination and concurrent clozapine. Clozapine itself tended to immediately lower circulating NK cell levels followed by a progressive marked increase following vaccination which remained increased even to Day 63. NK cells are thought to be involved in antibody isotype switching in the context of T-cell-independent type 2 antibody responses (Szomolanyi-Tsuda et al., 2001). Whether the progressive increase in NK cells represents a compensatory response that counteracted any direct effect of clozapine on anti-Vi antibody responses is unclear but NK cells are known to play important roles in the generation of long-lived memory and in the regulation of adaptive immune responses (Rydyznski and Waggoner, 2015). Specifically, NK cells have been shown to both promote (e.g. enhancing of antibody production and isotype switching) and suppress B cell function (e.g. impaired development of CD4+ T follicular helper cells and interference with germinal centre formation) dependent upon context (Rydyznski and Waggoner, 2015). Notably, NK cells have been reported to play a major role in stimulating T-l type 2 immune responses via release of IFN-y (Buchanan et al 1998), raising the possibility that their progressive induction in the active may have potentiated anti-Vi IgG responses (and/or counteracted other effects of clozapine to reduce this).

Together with the B cell findings described below, these findings suggest that clozapine exerts complex immunomodulatory effects beyond a discrete action on B cells or likely the adaptive immune system alone.

Clozapine has a broad impact on a range of B cell subsets, particularly memory pool cells

Low-dose clozapine administration to healthy subjects was associated with a rapid (i.e. days and before vaccination) reduction in mean circulating levels of almost all B cell subsets evaluated, particularly multiple distinct memory B cell pools. Memory B cells refer to cells that have encountered and responded to antigen and which have returned to a quiescent state and which remain responsive to a second challenge with antigen (Weisel and Shlomchik, 2017). These B cell subsets included:

• CD21- CD38 lo B cells (CD19+ CD21- CD38 low): CD21 (complement receptor type 2) is part of a complex acting as co-receptor to the B cell receptor (BCR) and variably expressed on B cells dependent on maturation stage (Thorarinsdottir et al., 2015). While most circulating B cells express surface CD21, including naive and memory cells, early transitional (Tl) B cells have low expression of CD21 while plasmablasts and plasma cells lack CD21 or express low levels. In addition, a discrete CD21-/low B cell subset has been described which is distinct to transitional and antibody producing cells and which are often expanded in patients with CVID (Thorarinsdottir et al., 2015).

CD21-low B cells have been reported to represent ~5% of peripheral blood B cells in normal adults and thought to be a form of memory B cell (Thorarinsdottir et al., 2016). They include CD38- CD24+ and CD38- CD24 low cells, each representing mostly CD27+ lgM+ lgD+ or switched CD27- B cells respectively. Analogous to classical memory B cells, they display markers indicating previous activation (i.e. antigen experienced cells) and they can proliferate and differentiate into plasmablasts (Thorarinsdottir et al., 2016).

Notably the frequency of CD21-/low B cells is expanded in multiple autoimmune disorders, including rheumatoid arthritis, SLE, Sjogren's syndrome as well as hepatitis C viral-associated autoimmunity, with a majority of these cells expressing germline autoreactive antibodies indicating autoreactivity) (Isnardi et al., 2010; Saadoun et al., 2013; Terrier et al., 2011; Wehr et al., 2004). • Class non-switched memory B cells (CD19+ CD27+ CD38lo lgM+): These will include lgD+ lgM+ unswitched memory B cells thought to represent circulating marginal zone (MZ) B cells (with a distinctive IgM hi subset bearing characteristics similar to MZ B cells that have recently passed through germinal centres) as well as IgD- lgM+ pre-switched memory B cells (Bautista et al 2020; Sanz et al., 2019).

Notably, CD19+ lgD+ CD27+ nonswitched memory B cells are reported to be activated in SLE, enriched for autoreactivity and possess a heightened capacity to migrate and interact with T cells (Rodriguez-Bayona et al., 2010).

• Class-switched memory B cells (CD19+ CD27+ CD38lo IgM- IgD-): these will include pre

existing memory reservoir (switched resting) or effector memory plasmablast/plasma cell precursor (i.e. switched activated) B cells expressing IgG, IgA or IgE. Class-switched memory B cells are known to be able to form robust secondary germinal centres during the recall response to antigen, with evidence of memory BCR diversification of class-switched secondary germinal centre B cells (McHeyzer-Williams et al., 2015). Indeed, class-switched memory B cells have been shown to be the dominant antigen-specific precursor cells for secondary germinal centres.

Notably, class-switched memory B cells are increased in autoimmune disease, such as lgG4- related disease, and also shown to be recruited to the cerebrospinal fluid during

neuroinflammation (Cepok et al., 2006; Kubo et al., 2018). As an early repopulating subset of memory B cells, have been used as a biomarker of the risk of disease re-activation in autoimmune disease, e.g. in the judging the re-treatment interval of rituximab in neuromyelitis optica spectrum disorder (dosing when these exceeded a threshold of 0.0005 x 10 ^A 9/L) (Trewin et al., 2020).

• lgD+ CD27+ ' innate like' memory B cells (CD19+ lgD+ CD27+): This B cell subset is known to predominantly produce IgM and contains two distinct memory B cell pools: both lgM+ (unswitched memory B cells) and IgM negative cells (i.e. IgD-only switched memory B cells whose biology is not well understood) (Agematsu et al., 1997). lgD+ CD27+ B cells do not appear to require classical germinal centres or conventional T cell help for their generation, given that they are present in individuals with X-linked hyperlgM syndrome characterised by absence of germinal centres and class-switched memory B cells. They are thought to be mobilised from the marginal zone in response to T-independent challenge (Wu et al., 2011). lgM+ lgD+ CD27+ lymphocytes ('IgM memory B cells') represent a large subpopulation of the human B-cell pool, possess typical memory B cell expression patterns (but distinct to IgM- only and lgG+ memory B cells), have a tendency to migrate to B cell follicles and re-enter (i.e. undergo secondary) germinal centre (GC) reactions upon reactivation, can undergo somatic hypermutation to generate high affinity lgM+ memory B cells and can differentiate rapidly into plasma cells upon interaction with activated neutrophils (Dogan et al., 2009;

Hara et al., 2015; Seifert et al., 2015). IgM hi IgD lo memory (CD19+ CD27+ CD38lo lgM+ IgD- ): These are likely to substantially represent pre-switched memory B cells, also known as 'IgM only memory B cells'. The findings are discussed more below.

• Circulating MZ-iike B cells (CD19+ CD27+ CD38+ lgM+ lgD+): Human splenic MZ cells have been described to have an IgM hi IgD low CDlc+ CD21 hi CD23- CD27+ phenotype and function as innate-like lymphocytes which interact with blood borne antigen to provide a first line of defence (Cerutti et al., 2013).

MZ B cells are able to rapidly differentiate into plasmablasts and mount rapid low affinity antibody responses to T cell-dependent and T cell independent antigens. They are usually associated with potent IgM responses, reflecting a lower activation threshold than follicular B cells, but can also class switch to produce IgG or IgA. Recently, IgM hi IgD low CDlc+ CD21 hi CD23- CD27+ B cells have been suggested to represent a heterogeneous population constituting both MZ B cells developing outside the germinal centre and unswitched memory B cells which develop in the germinal centre. MZ-like B cells have been observed as part of lymphocytic infiltrates in diseased autoimmune tissue, such as salivary glands from patients with Sjogren's disease (Daridon et al., 2006).

• Naive B cells (CD19+ CD27- lgM+ lgD+): These B cells have not been exposed to antigen can differentiate to both short-lived antibody-producing cells and seed germinal centre reactions. They respond less vigorously and rapidly to cognate antigen compared to memory B cells and form ~70% of blood B cells in humans (Deenick et al., 2013).

Defective B cell tolerance checkpoints in autoimmune disease, such as multiple sclerosis, are associated with an expansion of autoreactive naive B cells which may promote

autoimmunity through presentation of self-antigen to T cells (Meffre and O'Connor, 2019).

In particular, SLE is associated with an expansion of activated naive B cells which act as a source of antibody secreting cells and serum autoantibodies during disease flares (Tipton et al., 2015).

Plasmablasts (CD19+ CD27+ CD38+ IgM- IgD-): these are proliferative antibody-secreting cells derived from either activated naive or memory B cells. They are detectable at only low levels in health but are increased in a number of autoimmune diseases, e.g. lgG4-related disease, SLE, neuromyelitis optica spectrum disorder, where they may play a pathogenic role through production of autoantibody and cytokines (e.g. IL-6) and interaction with/antigen presentation to T cells, including induction of T follicular cell differentiation within germinal centres (Chavele et al., 2015; Lanzillotta et al., 2017).

• Transitional B cells (CD19+ CD27- CD24++ CD38++ lgM+ lgD+): Newly formed immature B cells in the bone marrow proceed to differentiate into transitional cell precursors which migrate into secondary lymphoid organs and can then differentiate into either follicular B cells or MZ B cells.

Transitional cells have been found in infiltrates of salivary glands of patients with Sjogren's syndrome, with TLR9 stimulation inducing proliferation and maturation into MZ-like cells, terminal differentiation into antibody secreting cells and autoantibody production (Guerrier et al., 2012).

The remarkable consistency of these effects across B cell subsets of different lineages and their temporal evolution compared with T cell compartment changes, is suggestive of a direct effect of clozapine on B cell biology and homeostasis. Further evidence supporting pharmacological effects of clozapine on B cells was the finding of significant consistently negative correlations between plasma clozapine level and circulating levels of B cell subsets including CD19+ B cells (see Figure 48), CD21- CD38lo, class-switched memory, lgD+ CD27+ (see Figure 49), and IgM hi IgD lo memory B cells. Notably, for several of these, there was a suggestion of reduction from baseline at a threshold effect at or above a plasma clozapine level of ~200 pg/L.

Clozapine leads to a rapid and near-complete elimination of pre-switched memory/lgM-only germinal centre-derived memory B cells (CD19+ CD27+ CD38lo lgM+ IgD-) from peripheral blood:

A striking finding of the study was the observation of rapid and near complete elimination of 'IgM hi IgD lo memory' B cells from the circulating compartment which commenced within the uptitration period (i.e. independent of vaccination) to reach a daily dose of 100 mg clozapine (see Figure 50). A key question emerges as to what this B cell subset then represents. Cells bearing a related surface immunophentoype: IgM hi IgD low CDlc+ CD21 hi CD23- CD27+ are regarded as heterogenous and proposed to include MZ B cellls developing outwith the germinal centre and 'unswitched' memory B cells developing inside the germinal centre (Cerutti et al., 2013). Flowever, the latter represent lgD+ lgM+ memory B cells are present in patients with CD40L-deficient hyperlgM syndrome unable to form germinal centres, suggesting a different origin for these pool of memory cells to pre-switched memory B cells (IgD- lgM+, i.e. IgM only) (Sanz et al., 2019). The reduced memory cell compartment observed in this study was lgM+ IgD- consistent with a 'pre-switched memory B cell' population, also known as IgM only memory B cells, although some degree of overlap with (i.e. partial inclusion of) an IgD lo population during the flow cytometry analysis is possible.

IgM-only memory B cells are thought to be generated in the early phases of a primary germinal centre reaction and exist in a pre-switched state capable of undergoing subsequent class-switching to any isotype, able to undergo subsequent rounds of germinal centre reactions to generate affinity matured isotype-switched B cells (Berkowska et al., 2011). IgM-only memory B cells have been shown to share a gene expression profile similar to that of post-germinal centre lgG+ memory B cells, with similar SMH levels in rearranged IGHV genes as germinal centre B cells, suggesting that these cells are early emigrants form the germinal centre that did not undergo class switching (Berkowska et al., 2011). Notably IgM-only and not MZ B cells, show a precursor-product relationship with switched memory (IgG or IgA) B cells (Bagnara et al., 2015; Palm and Henry, 2019). Notably lgM+ memory B cells have been shown to demonstrate marked stability compared with switched memory B cells, and through their ability to form germinal centres are thought to have the potential to become reservoirs of humoral memory over time for some subunit vaccines (Pape et al., 2011).

This study suggests a potent and specific impact on the IgM-only memory B cell subset, representing the first memory layer generated from naive B cells - whether this reflects a direct impact by clozapine to eliminate these cells, reduce their generation (i.e. inhibit early germinal centre formation) and/or sequester them in tissue/alter homing properties, is unclear from the current study. However, the abundance of these cells returned to baseline levels in the active arm in the post-dosing phase (i.e. by Day 63) (see Figure 50). Notably patients with CVID lacking IgM memory B cells have been shown to fail to produce anti-pneumococcal polysaccharide IgM and are at higher risk of pneumonia (Carsetti et al., 2005). Strikingly, long-term use of clozapine in patients with schizophrenia has been associated with reduced levels of pneumococcal-specific IgM, consistent with the current's study's findings of a potent inhibitory influence of clozapine on IgM-only memory B cell numbers and/or function (Ponsford et al., 2018b).

As a reservoir of antigen-experienced B cells, the potential therapeutic relevance of targeting this memory B cell pool, regarded as more stable over time than its class-switched counterpart (Roco et al., 2019), is supported by the observation of increased frequency of IgD- lgM+ memory B cells in patients with active SLE (Tipton et al., 2015). Notably, IgM autoantibodies are also known to result in accelerated SLE-like disease in murine models (Chevrier et al., 2014; Phan, 2014). Furthermore, recent observations highlight that the presence of a reservoir of IgM memory B cells may act as a continual source of pathogenic autoantibodies, such as in SLE, upon interaction with self -antigen (Roco et al., 2019).

Example 7

Keyhole Limpet Haemocyanin (KLH) T cell-dependent antibody response (TDAR) model study - effect of clozapine and norclozapine

Introduction

The T cell-dependent antibody response (TDAR) to antigen is an integrated measure of immune function that evaluates the kinetics and magnitude of an antigen-specific antibody (i.e. humoral) response following immunisation with a T cell-dependent antigen. TDAR is dependent upon the coordinated interaction and response of multiple immune cell types, including antigen presenting cells (primarily dendritic cells/macrophages), naive and activated CD4+ T cells (including T follicular helper cells, T _F H), B cells and antibody producing cells (plasmablasts and/or plasma cells). Functions evaluated by TDAR include antigen capture, processing (peptides on surface of MHC), cellular migration (e.g. of antigen presenting cell to draining lymph node or spleen), presentation to specific T/TFH cells, interaction of the latter with cognate B cells, cytokine & interleukin generation, germinal centre formation, somatic hypermutation, immunoglobulin class switching (e.g. of IgM to IgG), cellular proliferation & differentiation and antibody secretion. The TDAR assay can be used to measure the primary response (i.e. to a neoantigen) and/or secondary response (i.e. following repeated immunisation, reflecting memory B cell responses) to antigen (Lebrec et al., 2014; Plitnick and Herzyk, 2010).

Keyhole limpet haemocyanin (KLH) is a highly immunogenic protein derived from the haemolymph of Megathura crenulate and is used as a T cell dependent antigen. KLH is used extensively for TDAR evaluation (both primary & secondary immune responses), e.g. for pre-clinical immunotoxicology evaluation (i.e. unintended immunosuppression) and clinical studies (e.g. to measure primary and/or recall T cell-dependent specific antibody responses to novel immunotherapeutics in healthy volunteers and/or patients with autoimmune disease) (Abrams et al., 1999; Espie et al., 2020; Ferbas et al., 2013).

The objective of this experiment was to evaluate the ability of clozapine and norclozapine (NDMC) to impact on the T cell-dependent antibody response to a model antigen, KLH, including both primary immune and memory responses and to evaluate the cellular contributors to this. Method

Animals:

Young adult (age 6-7 weeks) C57BL/6 male mice were used for this study (Jackson Laboratories). The mice were housed at 21°C in individually ventilated cages (five mice per cage) with free access to food and water and a 12h light/dark cycle, Mice were acclimatised for at least a week prior to study start.

Compounds Clozapine and norclozapine (NDMC) were obtained commercially from Sigma-Aldrich Corporation. The CsA was made by Teva NDC 0172-7313-20 lot 100006032. CsA was in an alcohol solution (100 mg/ml) which was diluted out in 1% CMC/0.5% Tween 80.

Experimental groups, dose selection and immunisation: Mice were allocated into one of six experimental groups as follows:

1. Control vehicle (saline, IP, QD)

2. Clozapine 10 mg/kg (IP, QD)

3. Clozapine 25 mg/kg (IP, QD)

4. Norclozapine (NDMC) 25mg/kg (IP, QD) 5. Cyclosporin A (CsA) 50mg/kg (PO, BID)

Mice in group 1 (n=15) were treated by once daily IP injection of vehicle from Day 0 (day of immunisation) until the end of the study (Day 28).

Mice in groups 2-4 (n=15/group) were treated, after a short initial uptitration period, by once daily IP injection from Day -3 (before immunisation) until Day 28. Mice in group 5 (n=15) were treated by twice daily oral administration of CsA from Day 0 until Day 28.

On study Day 0 (after dosing of compound or vehicle) all mice were sensitised intradermally at the base of the tail with 0.1ml of lmg/kg Keyhole Limpet Haemocyanin (KLH)-TNP (i.e. Trinitrophenyl hapten conjugated to KLH protein) in Complete Freund's Adjuvant. On Day 14, mice remaining in the study (10 per group) were boosted intradermally with the same dose of KLFI-TNP (i.e. antigenic re challenge).

Biological samples for immunophenotyping:

On study days 7, 14 and 21 mice remaining in the study to Day 28 had samples taken via retro-orbital bleed under inhaled isoflurane anaesthesia. Similarly, on Days 14 and 28 (end of study), mice for tissue harvest were anaesthetised with inhaled isoflurane, bled by cardiac puncture and euthanised.

Serum was separated from all blood samples obtained and stored at -80°C for subsequent measurement of specific antibodies: anti-TNP IgM and anti-TNP IgG by ELISA.

On Day 14 (five mice per group) and on Day 28 (five mice per group) and following termination, tissue samples were rapidly collected from bone marrow (femur) and spleen and processed for evaluation of cellular composition in these compartments using multi-laser flow cytometric detection and analysis (FACS).

Individual antibodies employed for flow cytometry panels were pilot tested in the relevant tissues and the optimal dilution of each antibody determined to enable clear identification of

subpopulations. Flow cytometry data were acquired on a Beckman Coulter Cytoflex and analysed using the Beckman Coulter Kaluza software.

Statistical Analysis:

Data were analyzed by one-way ANOVA or using a mixed effects model with Flolm-Sidak's multiple comparisons test as appropriate. All calculations were made using GraphPad Prism software. A P value less than 0.05 was considered significant.

Results

Effects of compounds on spleen weight

At Day 14 post-initial KLFI-TNP challenge, the average absolute spleen weight in the control mice treated with vehicle was 0.109g (equivalent to a relative spleen weight of 0.398 when normalised to body weight, i.e. spleen weight [g] x body weight [g] x 100%). In a separate cohort of vehicle treated mice, absolute spleen weight was 0.121g (relative spleen weight 0.433) at Day 28 after challenge, an increase of 8.8% based on normalised splenic weight (Figure 51). At Day 14, both doses of clozapine were associated with significantly lower (P<0.05) absolute spleen weight compared to vehicle control (18.5 and 23.1% for 10 and 25mg/kg respectively) ( see Figure 51A). Neither norclozapine (NDMC) or the positive control, CsA, had a significant effect on either absolute or normalised splenic weight compared to vehicle control at this time point, although there was a numerically lower mean absolute weight in the NDMC group versus vehicle (11.8%). Indeed, clozapine dosed at 25 mg/kg led to a significantly lower absolute spleen weight at Day 14 compared to the reference positive control, CsA (see Figure 51A).

At Day 28, representing a timepoint 2 weeks post-repeat KLFI-TNP challenge, clozapine-treated mice exhibited substantial reductions in absolute spleen weight compared to vehicle-treated mice (P<0.0001), as well as significant reductions compared to mice treated with NDMC and CsA (at the higher dose of clozapine) ( see Figure 51C). Mice treated with NDMC also exhibited highly significant (P<0.0001) reduction in absolute spleen weight versus vehicle. There was no significant difference in mean absolute splenic weight between NDMC and CsA treated mice at Day 28.

A very similar pattern of results was observed with normalised spleen weights at Day 28. In particular, the normalised splenic weight of mice in both clozapine-treated groups was lower than in any other group, including very significant reductions compared to vehicle (P<0.0001), significantly lower than CsA (P<0.05), with a trend (P=0.05) to lower normalised weight than the NDMC group also ( see Figure 51D). All treatment groups showed significantly lower normalised splenic weight compared to vehicle-treated mice. The positive control compound CsA (dosed at 50mg/kg PO BID) had no discernible effect on spleen weight at Day 14 (+1.0%) but there was a significant reduction at Day 28 versus vehicle control (16.1%), although this was significantly (P<0.05) less marked than found in mice treated with either dose of clozapine.

Effects of compounds on primary and secondary T cell-dependent antibody responses KLFI-TNP Primary acute IgM response to KLH

Primary immunisation with KLFI-TNP induced a robust acute induction in serum anti-TNP specific IgM in the vehicle treated group measured as 45,397 U/mL at Day 7 (Figure 52). Administration of clozapine at 25 mg/kg exerted a strong trend (P=0.07) when corrected for multiple comparisons, or significant reduction (P=0.03) using an uncorrected Fisher's LSD test, to lower TNP-specific IgM by ~30% to a mean value of 31,841 U/mL at this time. Norclozapine, had no significant effect on anti- TNP IgM levels at Day 7.

The positive control compound CsA markedly reduced anti-TNP IgM levels at Days 7 (by 87.7%. As expected, the magnitude of primary response was greater with anti-TNP IgM than IgG also measured at Day 7 (see Figure 53B).

Specific IgG response to KLH-TNP immunisation

The overall kinetics of specific IgG response to KLH-TNP primary immunisation at Day 1 and re challenge at Day 14 are shown in Figure 53A. Highly significant effects of treatment group and time were seen on anti-TNP IgG with lower peak anti-TNP peak IgG at Day 28 in both clozapine-treated cohorts than in any other group. These findings are discussed further below.

Primary IgG response to KLH

Primary immunisation with KLH-TNP induced a robust induction in TNP-specific IgG in serum of vehicle treated mice, measured as 23,120 U/ml at Day 7, increasing sharply further to 389,027 at Day 14 following initial challenge (measured before re-challenge) (see Figure 53B and 53C).

Lower dose clozapine resulted in a highly significant (P<0.0001) reduction in the primary IgG response to KLH-TNP that was apparent by Day 14 (by 57% vs vehicle treated control).

The higher dose of clozapine (25mg/kg) produced significant reductions in anti-TNP IgG at Day 7 (63.3%) and Day 14 (57.8%) consistent with an early potent impact on B cell class-switching. The level of anti-TNP IgG was numerically higher although not statistically significantly different to mice treated with the positive control agent, CsA, at both Day 7 and Day 14.

Norclozapine (25mg/kg) had no significant effect on anti-TNP IgG levels at Day 7 but by Day 14 had a highly significant (P<0.001) effect to suppress primary IgG response (by 46.1% vs vehicle), although the magnitude of reduction was marginally lower than with either dose of clozapine at this time point.

The positive control compound CsA exerted highly significant reductions in primary IgG response to KLH-TNP at both Day 7 and Day 14 (by 80.6% vs vehicle). The levels of anti-TNP IgG induced at Day 7 and 14 in response to CsA were not significantly different to those observed in mice treated with higher dose clozapine.

Secondary IgG response to KLH

The secondary antibody response to KLH was measured as serum anti-TNP IgG at Day 21 and Day 28 (i.e. 7 and 14 days post-second immunisation with KLH-TNP, respectively) (see Figure 53D and 53E). Repeat immunisation with KLH-TNP induced a further robust induction in TNP-specific IgG in serum of vehicle treated mice at Day 21 and again at Day 28, reaching a mean of 630,929 U/mL and 1,065,485 U/mL, respectively.

Both doses of clozapine exerted significant and marked suppressive effects on this secondary (i.e. memory) response, with mean values of serum anti-TNP IgG of 297,259 and 237,304 U/mL at Day 21 in mice dosed with 10 mg/kg and 25 mg/kg clozapine, respectively (see Figure 53D). Similar findings were apparent at Day 28 (see Figure 53E).

Mice treated with NDMC or CsA both exhibited smaller but still significant (P<0.05) suppression of anti-TNP IgG at Day 21 (both ~39% vs vehicle). Strikingly, the magnitude of reduction in secondary IgG response was greater with either dose of clozapine than the positive control CsA, in part reflecting the marked rise in anti-TNP IgG with re-immunisation in CsA-treated mice suggesting less ability of the latter to impact upon memory responses.

Conclusion

This study evaluated the ability of clozapine and norclozapine (NDMC) to impact upon the widely used TDAR assay. The major findings are as follows: a) Clozapine induces a significant reduction in spleen weight of KLH-immunised mice and greater than that observed with a potent positive reference compound, ciclosporin A (CsA). b) Clozapine exerts a significant reduction in the acute IgM response to primary immunisation with KLH-TNP, although this is less than that of CsA. This reduction in acute specific IgM was not observed with norclozapine. c) Clozapine exerts potent suppressive effects on the primary IgG response to KLH immunisation, with greatest reductions observed with the higher dose employed. d) The reduction in primary IgG antibody response is (by Day 14) comparable to that observed with the positive control, a high dose of CsA. e) Clozapine at both doses substantially suppressed the secondary antibody response to KLH consistent with a strong ability to dampen B cell memory responses. f) The suppression in secondary IgG antibody response induced by clozapine was greater than that achieved by CsA.

Taken together, these findings indicate that clozapine reduces both primary (IgM-dominated and early IgG response) and secondary humoral responses to immunisation with KLH (see Figure 53A), a T cell-dependent antigen, a process which involves antigen processing and presentation, priming and interaction between T and B cells, B cell activation, antibody generation by B cells and cytokine- dependent class-switch recombination.

Clozapine's ability to reduce the weight of the spleen - the largest lymphoid tissue in mammals - is a clear indication of immune system effects.

The initial wave of antibody generation after exposure to a T-dependent antigen reflects migration of activated B cells to extrafollicular foci to undergo plasma cell differentiation, generally as short lived plasmablasts producing low-affinity immunoglobulin (Paus et al., 2006). Subsequently, after approximately 1 week, a second wave of T cell-dependent plasma cell production derives from antigen-primed B cells which enter the primary follicle and propagate the germinal centre reaction to undergo antigen-specific B cell selection, expansion, affinity maturation via somatic

hypermutation and class-switching to generate memory B cells and long-lived plasma cells

(Quemeneur et al., 2008). Accordingly, the findings of reduced anti-TNP IgG at both Day 7 and Day 14 suggest that clozapine can inhibit both extrafollicular plasma cell and germinal centre pathways of B cell differentiation in response to a T-dependent antigen.

In mice, the peak IgM response to KLH is known to occur within the first week (White et al., 2007). While primary KLH immunisation is classically regarded as recruiting naive B cells only, findings from human studies indicate that primary KLH immunisation recruits both from the naive B cell repertoire and from cross-reactive memory B cells (Giesecke et al., 2018). Secondary immunisation antibody responses are thought to near-exclusively recruit from the memory repertoire, with minimal clonal overlap with the primary immune response. Accordingly, the prominent suppressive effect of clozapine and norclozapine on the secondary antibody response to KLH suggests a potential preferential ability to inhibit memory B cell activation and/or responses, of particular potential therapeutic relevance in the context of pathogenic immunoglobulin-driven autoimmune disorders.

Strikingly, clozapine and norclozapine exerted an equivalent or even greater impact on memory response than that achieved by CsA. The latter, used as a positive reference immunosuppressive compound in the study, is a highly potent inhibitor of T lymphocyte activation used clinically in organ transplantation and severe/refractory autoimmune disorders. In contrast, CsA exerted greater effects on the primary antibody response. These observations are in keeping with a comparable breadth of immunomodulatory potential of clozapine compared with this potent standard of care immunotherapeutic. Other immunosuppressive drugs used for the treatment of pathogenic immunoglobulin treated disorders also exhibit suppressive effects in the KLH TDAR assay, including corticosteroids, azathioprine, FK506 (tacrolimus) and cyclophosphamide (Gore et al., 2004; Kawai et al., 2013; Ulrich et al., 2004).

The translational relevance of suppressing antigen-specific responses is further highlighted by the use of KLH in multiple preclinical and clinical studies examining the potential of novel

immunomodulators to impact upon humoral immune activation in health and autoimmune disease (Karnell et al., 2019; Klimatcheva et al., 2015; Nicholson et al., 2020; Sullivan et al., 2016).

Example 8

In vitro B cell profiling of clozapine and norclozapine (NDMC) using human donor B cells

Introduction

Dysregulated B cell signalling is recognised as a primary driver of autoimmune disease; specifically, altered B cell intrinsic signals mediated via the B cell receptor (BCR) and key co-receptors (e.g. CD40, CD80 and CD86) can promote spontaneous autoimmunity. Altered B cell signalling is thought to facilitate a breach in B cell tolerance, impacting B cell selection during the establishment of the naive BCR repertoire (i.e. skewing the pre-immune repertoire toward greater baseline poly- and self reactivity) and can affect extra-follicular and germinal centre pathways of B cell activation to lead to the generation of autoantibody producing B cells (Rawlings et al., 2017).

The BCR acts as a master regulator of central and peripheral tolerance mechanisms during B cell development and maturation (Rawlings et al., 2017). These tolerance mechanisms are required given the inherent randomness in the process of generating a large repertoire of antigen-specific receptors leading to the generation of self-reactive B cells. Thus, negative selection of self-reactive B cells (via mechanisms including cell deletion, receptor editing or induction of anergy) are largely mediated via BCR signalling, with input from Toll-like receptors (TLRs). Positive selection occurs largely in the periphery in transitional B cells (e.g. via survival and/or clonal expansion) and is driven by an interaction between BCR signalling and co-receptor signalling (e.g. BAFFR, CD40, TLRs).

Substantiating the importance of the BCR, multiple polymorphic risk loci involving genes encoding proteins involved in or downstream of BCR signalling have been associated with human autoimmune disease, including BANK1 (scaffold protein involved in BCR signalling), BLK (B lymphoid tyrosine kinase, a B cell linker protein highly restricted to B cells), ITGAM (integrin alpha M or CDllb, a negative regulator of BCR signalling), LYN (tyrosine kinases) and RASGRP3 (encoding GRP3, involved in signalling downstream of the BCR) (Ding et al., 2013; Flan et al., 2009; Flom et al., 2008; Taher et al., 2017). Functional studies of B cells upon BCR activation from patients with SLE have revealed defective signalling (Flores-Borja et al., 2007), including evidence of continual activation via the BCR without appropriate T cell-derived co-stimulation and enhanced survival of lupus B cells and differentiation into plasma cells (Fleischer et al., 2016; Vasquez et al., 2019; WeiRenberg et al., 2019). Defective B cell responses have also been identified in several other autoimmune disorders, including systemic sclerosis, immune thrombocytopenia and Sjogren's syndrome (Corneth et al., 2017; Forestier et al., 2018; Taher et al., 2018; Wang et al., 2018).

Accordingly, B cell profiling was undertaken to evaluate the impact of clozapine and norclozapine on early signalling downstream of BCR engagement, as well as the impact of these molecules on the activation, proliferation and differentiation of human B cells.

Method

Two experiments were performed using total peripheral blood B-cells isolated from six independent healthy donors. Both experiments utilized the same donor samples.

Compounds

Compounds were obtained commercially from Sigma-Aldrich Corporation and dissolved in DMSO. They were diluted with (media) to the final concentrations (below) used in the experiments.

Experiment 1

Human B cells were purified from peripheral blood mononuclear cells (PBMCs) from six healthy donors and then stimulated with soluble anti-lgM or vehicle (control) following pre-incubation with clozapine or norclozapine (NDMC) (two test compounds each at the following final concentrations; 10 ng/ml, 30 ng/ml, 100 ng/ml, 300 ng/ml, 1.0 pg/ml and 3.0 pg/ml; preincubation for 60 minutes), reference compound 1 (Ibrutinib at four concentrations; 300nM, luM, 3uM and lOuM;

preincubation for 60 minutes); reference compound 2 (Syk inhibitor BAY-61-3606 at four concentrations; 300nM, luM, 3uM and lOuM; pre-incubation for 60 minutes) or a vehicle (0.1% DMSO) control.

Stimulated B cells were immuno-stained after 5 minutes with the following panels quantified by flow cytometry to identify signalling molecule changes in the major peripheral B-cell subsets including naive, 'B , 'B2', transitional, and memory populations.

Calcium signalling was analysed by flow cytometry. B cells isolated from PBMC were incubated for 1 hour at 37C with Fluo-4 in the presence of clozapine, norclozapine (NDMC), ibrutinib or BAY-61-3606 in the absence (vehicle [0.1% DMSO]) or media alone at the indicated concentrations. After establishing a 20 second baseline on the Flow Cytometer, cells were treated with vehicle or anti-lgM (10 pg/ml) and recorded for a further 180 seconds.

Experiment 2

B cells from the same donor were stimulated with either: (i) vehicle (control; 0.1%DMSO); (ii) soluble anti-lgs + recombinant CD40L + IL-21 ('T cell-dependent' -stimulation mimic); (iii) anti-lg presented in polymeric form ( -cell independent type 2' -stimulation mimic); or (iv) CpG+ IL-15 ([BCR

independent] 'T cell-independent'-stimulation mimic); or following pre-incubation with clozapine or norclozapine (each at the following final concentrations; 10 ng/ml, 30 ng/ml, 100 ng/ml, 300 ng/ml, 1.0 pg/ml and 3.0 pg/ml), reference compound 1 (Ibrutinib, a BTK inhibitor, at four concentrations; 300nM, luM, 3uM and lOuM; pre-incubation for 60 minutes), reference compound 2 (BAY 61-3606, a Syk inhibitor, at four concentrations; 300nM, luM, 3uM and lOuM; pre-incubation for 60 minutes), or reference compound 3 (chloroquine, a TLR inhibitor, at four concentrations; 3uM, lOuM, 30uM and lOOuM; pre-incubation for 60 minutes) and vehicle control.

After five days of culture, the following readouts were performed: i. Cell proliferation assessed by flow cytometry (by measuring dilution of eFluor450 staining), absolute cell counts (using counting beads) and cell viability. ii. Immuno-labelling of B cell activation markers was be quantified by flow cytometry measuring activation marker expression (CD71, CD80, CD86). iii. Cytokine levels (IL-6, IL-8, IL-10, IL-12, TNFa, IFNg) were measured in the cell culture supernatant by multiplex analysis (Luminex; single dilution of cell culture supernatant).

Results

Effects of compounds on Ca ²⁺ signalling following anti-lgM stimulation

The change in intracellular Ca2+ levels in total B cells on the addition of anti-lgM and the impact of pre-incubation with clozapine, norclozapine (NDMC) and reference compounds (Ibrutinib and Bay- 61-3606) averaged across 6 donors is shown in Figure 54 and represented as: (A) peak response; (B) Area under the Curve (AUC); (C) % responding cells and (D) slope of response

There was a clear anti-lgM-invoked Ca2+ response seen in all six donors for all parameters measured (see Figure 54). There were between 20-50% of responding cells across the donors with an average of around 30% within 3min of anti-lgM addition. There was some indication of a bimodal impact of clozapine and norclozapine on the anti-lgM invoked Ca2+ response across the different parameters in as much as low concentrations (0.01-0.03 pg/mL) showed a tendency to increase the anti-lgM-invoked Ca2+ response while concentrations 1- 10pg/mL clearly inhibited the response to below control levels of anti-lgM-induced stimulation. This apparent bimodality was particularly marked when scrutinising the Ca2+ response as the slope, effectively a measure of the kinetics of Ca2+ mobilisation in response to anti-lgM. In the one donor where the concentration of clozapine and norclozapine was increased to lOug/ml the measure of slope was reduced to baseline (for both compounds) with the other parameters falling well below that of control anti-lgM-stimulated levels (see Figure 54D).

With respect to the inhibitory element of the response, clozapine appeared somewhat more potent than norclozapine, a finding consistent across all six donors.

With regards to the reference compounds Ibrutinib returned the anti-lgM invoked Ca2+ response to near basal levels at all concentrations tested (0.3-10uM), while Bay-61-3606 (0.3-10uM) showed a partial concentration-dependent inhibition of the response. Notably, at the higher concentration(s), clozapine and norclozapine had a largely comparable inhibitory effect on peak Ca2+ response and AUC to that of the reference compounds. With respect to the measure of slope, higher dose clozapine and norclozapine induced greater reduction than all doses tested of either ibrutinib or (even more pronounced) than the Syk inhibitor (see Figure 54D).

Impact of clozapine and norclozapine on B cell activation markers induced in response to T cell- dependent and T cell independent stimuli

The change in cell surface activation markers CD71, CD80 and CD86 in B Cells following a 5-day culture with different stimuli and the impact of clozapine, norclozapine and reference compounds (Ibrutinib and Bay-61-3606) versus unstimulated cultures is shown in Figure 55A-C (reflecting averaged data from 6 donors). The stimuli used were: (i) anti-lg/CD40/IL-21; (ii) polymeric anti-lgM; and (iii) CpG/IL-15.

Incubation of the B cell cultures with either anti-IG/CD40/IL-21 or CpG/IL-15 resulted in up- regulation of all three B cell activation markers over unstimulated (control) cultures (see Figure 55A- C). Culture with polymeric anti-lgM led to a selective up-regulation of CD86 (see Figure 55A-C).

Clozapine and norclozapine at 3pg/mL produced partial and variable inhibition of the up-regulation of all the activation markers, depending on the stimulus used and the marker being scrutinised. CD71 and CD80 up-regulation was inhibited by >50% in anti-lg/CD40/IL-21 cultures more or less equipotently by both clozapine and norclozapine, whereas in CpG/IL-15 cultures norclozapine appeared to be superior to clozapine (see Figure 55A-B).

The inhibition of CD86 up-regulation under all three stimulating culture conditions was modest, the most pronounced inhibition being noted in CpG/IL-15 cultures with norclozapine (see Figure 55C). In the one donor where the concentration of clozapine and norclozapine was increased to lOpg/mL the CpG/IL-15 induced up-regulation of all three activation markers was almost completely inhibited, as was that of CD86 in polymeric anti-lgM cultures, but less so of activation markers in anti- lg/CD40L/IL-21 cultures. Notably, clozapine and norclozapine also inhibited proliferation of B cells in response to CpG-ODN/IL-15 stimulation (data not shown).

The three reference compounds demonstrated variable degrees of concentration-dependent inhibition of activation marker up-regulation depending upon mode of stimulation and the marker being scrutinised (see Figure 55).

Effects of clozapine and norclozapine on T-cell dependent and T cell independent increases in B cell cytokine production

The donor average data for supernatant cytokines IL-6 and TNF-a from 5-day cultures of purified B- cells stimulated as described above are shown in Figure 56 and Figure 57.

Anti-lg/CD40/IL-21 and CpG/IL-15 both promoted the release of IL-6 and TNFa markedly above that of control unstimulated cultures (the latter having near undetectable cytokines levels, data not shown). Stimulation of these cytokines with polymeric anti-lgM was significantly lower in comparison.

Norclozapine produced a substantive and largely concentration-dependent inhibition of IL-6 and TNFa release from both the anti-lg/CD40/IL-21 and CpG/IL-15 cultures. Clozapine appeared less effective but demonstrated some partial inhibition of IL-6 and TNF-a stimulated release at 3pg/mL.

In the one donor where the concentrations of clozapine and norclozapine were increased to lOpg/mL, the inhibition of both IL-6 and TNF-a from cultures stimulated with either anti-lg/CD40/IL- 21 or CpG/IL-15 were again increased, consistent with a dose dependent suppression of the release of these cytokines by clozapine and norclozapine in response to the stimuli.

Although stimulation of IL-6 and TNF-a release by polymeric anti-lgM was low, there were dose- dependent inhibitions of release of these cytokines with both clozapine and norclozapine, with norclozapine again appearing the more potent. All three reference compounds demonstrated inhibition of induced IL-6 and TNF-a release, the level of which varied with stimulation condition.

Notably, the suppression of anti-lg/CD40/IL-21-stimulated B cell IL-6 release by norclozapine was comparable to that achieved by either the BTK or Syk inhibitor (both at 0.3 mM). Norclozapine at the higher concentration showed comparable efficacy in suppressing polymeric anti-lgM induced IL-6 release to that achieved by the Syk inhibitor. Norclozapine showed greater efficacy in suppression of IL-6 release by B cells cultured with CpG-ODN + IL-15, than either ibrutinib or the Syk inhibitor given at 10 pM. As expected, chloroquine was exceptionally potent under these culture conditions compared to the other B cell activation stimuli.

A similar pattern of comparable efficacy of norclozapine was observed for suppression of TNF-a release from B cells cultured in either anti-lg/CD40/IL-21 or CpG/IL-15 to that of the BTK and Syk inhibitors used at 0.3-1.0 pM (Figure 57).

Conclusion

This study evaluated the potential for clozapine and norclozapine to exert direct effects on fundamental aspects of B cell biology, including BCR-triggered Ca2+ responses, impact on upregulation of B cell activation markers induced by multiple mechanisms of B cell activation and associated release of pro-inflammatory cytokines. The major findings of this study are: a) Clozapine and norclozapine suppressed BCR-promoted Ca2+ responses at higher concentrations, consistent with an effect of these compounds on B cells to inhibit BCR signalling. b) Clozapine and norclozapine inhibit anti-lg/CD40-L/IL-21- and CpG/IL-15-stimulated expression of multiple B cell activation markers (CD71, CD80 and CD86), consistent with a direct inhibition of B cell activation by both these compounds. c) Clozapine and norclozapine exerted dose-dependent, functional inhibitory effects on B cell production of key cytokines implicated in the germinal centre response and/or inflammation: IL-6 and TNF-a. d) The limited ability of either clozapine or norclozapine to influence polymeric Ig-mediated B cell activation concurs with the finding of no impact on specific response to Typhi Vi immunisation in healthy volunteers (see Example 6). d) The above effects of clozapine and norclozapine at the higher concentrations tested are, in most cases comparable to or superior to those observed with positive reference control compounds inhibiting BTK or Syk which target fundamental downstream components of the BCR pathway and/or which are being used (or investigated) clinically for their potent effects on B cells. e) Together, these observations indicate that both clozapine and norclozapine can influence B cell intrinsic properties to inhibit multiple core pathways of B cell activation.

Engaging the IgM class of the B cell receptor (BCR) on B cells negatively isolated from healthy donors' peripheral blood with soluble F(ab')2 anti-lgM resulted in a robust and rapid increase in intracellular Ca2+ in approximately 50% of total B cells, as anticipated from the total frequency of lgM+ B cells in peripheral blood. While low doses of clozapine and norclozapine exhibited a tendency to enhance the anti-lgM stimulated Ca2+ response at low levels, higher concentrations led to substantial blunting of BCR-promoted Ca2+ responses. Upon BCR activation, BTK is activated by tyrosine protein kinases LYN and SYK (spleen tyrosine kinase), which is followed by 1- phosphatidylinositol-4, 5-biphosphate phosphodiesterase gamma-2 (PLCy2) activation which promotes the release of intracellular Ca2+ from the endoplasmic reticulum and store-operated Ca2+ influx (Kim, 2019). The BCR has two key interrelated functions: to initiate signalling cascades that lead to major changes in the actin cytoskeleton, metabolic remodelling and the expression of genes encoding proteins associated with B cell activation; and the internalisation of bound antigen for intracellular processing of antigen-derived peptides for presentation on MHC class II molecules to helper T cells (Kwak et al., 2019). BCR-dependent Ca2+ signalling has an important role in B cell development and fate, including contributing to self-tolerance mechanisms responsible for the elimination of autoreactive B cells. As a corollary, murine models of increased BCR signalling exhibit a greater prevalence of autoimmune disease (Dorner and Lipsky, 2006), while dysregulated Ca2+ signalling has been reported in several autoimmune disorders including SLE, rheumatoid arthritis and primary Sjogren's syndrome (Hasegawa et al., 2005; Hemon et al., 2017).

Both clozapine and norclozapine had specific ability to inhibit anti-lg/CD40-L/IL-21- and CpG/IL-15- driven B cell activation and associated cytokine release. These stimuli are designed to mimic T cell dependent and BCR-independent T cell-independent stimulation, respectively. In conjunction with the findings from the in vivo immunisation with KLH, the observation of reduced expression of B cell activation markers and cytokine release in response to anti-lg/CD40-L/IL-21 concordantly indicate that clozapine and norclozapine substantially inhibit T cell-dependent B cell activation. Specifically, the in vitro conditions utilised are widely accepted to mirror T helper support (in particular T follicular helper, Tfh, cell) involving CD40L interactions with CD40 expressed on the B cell and the cytokine IL-21, substantially expressed by Tfh cells, and a key regulator of B cell differentiation particularly in the germinal centre (Franke et al., 2020). These observations also provide an in vitro link to the clear signal for reduced germinal centre function with clozapine in the collagen-induced arthritis model. Importantly, ectopic germinal centre formation (also known as tertiary lymphoid structures) are a key component of multiple autoimmune disorders, structures whose formation can be IL-21 dependent (Deteix et al., 2010; Pipi et al., 2018).

The ability of clozapine and norclozapine to inhibit proliferation of B cells in response to CpG- ODN/IL-15 stimulation is of particular interest given the role self-containing immune complexes are thought to have in driving a TLR-dependent immune response. CpG-containing

oligodeoxynucleotides (CpG ODNs) act on Toll-like receptor 9 (TLR9) primarily expressed on B cells and dendritic cells. TLR9 is a nucleic-acid-sensing TLR recognising unmethylated DNA from bacteria or released by apoptotic cells and whose dysregulation is implicated in autoimmune disease and autoantibody production. Accordingly, loss of TLR function has been shown to improve multiple genetic models of SLE. TLR9 and the BCR function synergistically in B cells to enhance cell proliferation, cytokine and antibody production (Kim et al., 2009; Szili et al., 2014). As a corollary, suppression of TLR-9 and BCR-mediated activation by clozapine and norclozapine may have the potential to directly suppress pathogenic B cells in autoimmune disease.

Activated B cells are known to produce IL-6 which can promote Tfh cell differentiation and critically trigger spontaneous germinal centre formation leading to a break in B and T cell tolerance and the development of systemic autoimmunity (Arkatkar et al., 2017). Indeed, lack of B cell-derived IL-6 prevents spontaneous GC formation in murine SLE and protects against lupus nephritis, even in the presence of IL-6 production by non-B cells. Elevated IL-6 is known to drive inflammation in a broad range of autoimmune disorders and its levels correlate with disease activity, including in rheumatoid arthritis, SLE and multiple sclerosis. As a pro-inflammatory modulator of T cells, the ability of clozapine and norclozapine to suppress B cell derived IL-6 may interfere with the latter's effects to promote pro-inflammatory autoreactive CD4+ T cell responses (via enhancement of Thl7 cell function and suppression of Treg induction) (Jones et al., 2018). In addition, given that B cells from patients with autoimmune disease have been shown to produce more TNF-a than those of healthy donors, the ability of clozapine and norclozapine to reduce the latter is expected to be potentially beneficial.

Summary of Results set out in Examples 1-8:

The results set out in the examples above, encompassing observational data in humans treated with clozapine for prolonged periods of time, short term dosing in healthy wild type mice in an immunologically unchallenged setting, evaluation in a disease model of autoimmune disease with a major B cell component driven by antigen (CIA model), short-term low dose administration to healthy human volunteers as part of a primary vaccine challenge, primary and recall immunisation with a T cell dependent antigen in vivo using clozapine and norclozapine, and in vitro profiling of human donor B cells highlight several key effects of clozapine (and/or norclozapine where tested) on B cell and other immune biology:

1. Reduction in total circulating immunoglobulin levels affecting all classes evaluated (IgG, IgM and IgA). While exhibiting interindividual variation, clozapine is seen to result in a leftward shift in the frequency distribution curve for these immunoglobulins. The robustness of this finding is highlighted by the interim findings in an orthogonal cohort of patients taking clozapine or other antipsychotics.

2. A relatively greater impact in human to reduce IgA (and IgM) compared to IgG, in part recapitulated with short-term dosing of wild type mice.

3. Evidence of progressive immunoglobulin (IgG) reduction with increasing duration of clozapine exposure in human. Conversely, evidence of gradual recovery (over years) of IgG on clozapine cessation.

4. Reduction in specific immunoglobulin. Beyond reductions in total immunoglobulin titre, clozapine is seen to lower pathogenic immunoglobulin (CIA model) and has been demonstrated by the inventors to lower pneumococcal specific antibody in human (Ponsford et al., 2018b), with the latter demonstrating a strong trend to significantly lower values on even interim analysis of the second observational cohort.

5. No significant impact on overall circulating (CD19+) B cells numbers. This observation contrasts sharply with the impact of current aggressive generalised B cell depleting biological approaches.

6. Substantial reductions in circulating plasmablasts (short-lived proliferating antibody secreting cells of the B cell lineage) and class-switched memory B cells. Both cell types are critical in the immediate and secondary humoral response. Class-switching enables a B cell to switch from IgM to production of the secondary IgH isotype antibodies IgG, IgA or IgE with different effector functions (Chaudhuri and Alt, 2004). Increased class-switching and plasma cell differentiation is recognised as a key feature in autoimmune disease associated with pathogenic immunoglobulin production (Suurmond et al., 2018). An ability of clozapine to inhibit this process, i.e. reduce class-switched memory B cells, suggests particular therapeutic potential in the setting of pathogenic immunoglobulin-mediated disorders which are primarily mediated by autoantibodies of the IgG, IgA or IgE subclass. 7. Subtle effects on bone marrow B cell precursors, specifically including a reduction in total pre B cells, proliferating pre B cells and immature B cells. This is notable for being a key endogenous transition checkpoint of B cell development for autoreactivity (Melchers, 2015). Defective B cell tolerance, including early tolerance, is recognised as a fundamental feature predisposing to autoimmunity (Samuels et al., 2005a; Yurasov et al., 2005). Accordingly, while speculative, it is possible that this effect of clozapine will serve to reduce further progression of B cells with autoreactivity (of the IgH chain) to modulate the emerging B cell repertoire.

8. Reduction in bone marrow long-lived plasma cells, a key cell population responsible for driving persistent autoimmune disease through the production of pathogenic immunoglobulin and which is substantially refractory to existing therapeutics.

9. The ability to substantially delay the onset of an experimental model of autoimmune disease with a substantial B cell-driven and pathogenic autoantibody component.

10. Disruption of germinal centre function through effects on its key cellular components: induction of a profound reduction in germinal centre B cells together with a reduction their level of activation/functionality. Coupled with this, clozapine is found to reduce in surface expression of key proteins regulating T follicular helper cell positioning and functionality (PD1 and CXCR5). Germinal centres are the sites of intense proliferation and somatic mutation to result in differentiation of antigen-activated B cells into high affinity memory B cells or plasma cells. Accordingly, this finding (following antigen injection in the CIA model) is consistent with an impact of clozapine on distal B cell lineage maturation/function and modulation of T cell support of these processes. The net effect of this is concordant with observations set out in the examples demonstrating reduced class switched memory B cells, reduced plasmablast and long-lived plasma cell formation in response to clozapine. Together these actions will tend to reduce pathogenic immunoglobulin production in the setting of B cell driven autoimmune disease, including those with a T cell component.

11. Based on an in vitro differentiation assay, the observed effects of clozapine appear unlikely to reflect primarily a direct effect on (late differentiated) antibody secreting cells.

12. Reduce the proportion of B cells in secondary lymphoid tissue.

13. Promote a significant increase in the proportion of Foxp3 ⁺ regulatory T cells (Tregs) in secondary lymphoid tissue in conjunction with an increase in the expression of the Treg marker CD25 (IL-2 receptor a-chains). Tregs are a specialised CD4+ T cell subset with a major immunoregulatory role in promoting immune tolerance and actively suppressing autoimmunity. IL-2 signalling is critical to maintaining Treg homeostasis and CD25 has been proposed to be used by Tregs to capture IL-2, thereby limiting its provision to and stimulation of effector CD4 ⁺ T cells to promote the latter's apoptosis. Accordingly, higher cell surface expression intensity of CD25 may serve to promote immunosuppressive Treg function.

14. Short-term (~4 weeks) of low dose (100 mg) of clozapine to healthy volunteers receiving primary immunisation with Typhim Vi being associated with:

• Progressive reduction in total IgM.

• Transient reduction in total and anti-pneumococcal specific IgG at Day 7 post-vaccine.

• A rapid (<7 days) reduction below baseline of multiple circulating B cell subsets, including multiple different memory B cell pools of different lineages.

• Complete elimination of detectable pre-switched (IgM-only) memory B cells, early emigrants from primary germinal centre reactions which act as direct precursors to switched memory B cells. The observation of a robust impact on IgM-only memory B cells coupled with the known effects of clozapine to reduce class-switched memory B cells and reduce total immunoglobulins (IgM, IgG and IgA), is consistent with a primary action on the germinal centre, i.e. primarily an effect on T cell-dependent pathways of B cell activation and antibody generation.

• Significant consistently negative correlations between plasma clozapine levels and multiple B cell subsets, consistent with a direct pharmacological effect of clozapine on B cell biology apparent even at the low doses employed for study.

• No significant impact on induction of Vi-specific IgG (a T-independent type 2 antigen

response) suggesting that clozapine's action on B cells is likely to be mediated via other core mechanisms of B cell activation (specifically, T cell-dependent and/or T cell independent, type 1 responses).

• Leucocytosis and lymphocytosis, particularly affecting the CD4+ T cell compartment.

• Clozapine's specific effects on multiple memory B cell subtypes may be of therapeutic utility in autoimmune/other disorders driven by dysregulated and/or autoreactive B cells and/or germinal centres or related tertiary lymphoid structures.

15. Highly effective in suppressing primary and secondary IgG responses to a model T cell- dependent antigen, KLH-TNP, in vivo (TDAR model). Together with the other data, this suggests a potent influence of clozapine on primary germinal centre formation, secondary germinal centre reactions and associated processes of immunoglobulin class-switching, memory B cell formation and plasmablast/plasma cell production.

16. Suppresses acute primary IgM responses to a T cell dependent antigen in vivo, suggesting the ability of clozapine to also act on very early T cell dependent antibody production by extrafollicular- derived B cells before class-switching. This observation is congruent with the acute and prominent impact on the IgM only memory B cell population identified in the healthy volunteer study.

17. Clozapine's efficacy in the TDAR model is comparable or greater to that observed with high dose twice daily administration of ciclosporin A, a potent immunosuppressant acting to inhibit calcineurin primarily working on T helper cells and used clinically to reduce the risk of transplanted organ rejection, graft-versus-host disease, and severe active autoimmune disease. Similar findings were observed with norclozapine (NDMC).

18. Produces substantial reductions in splenic size in mice immunised with KLH-TNP and greater than that observed with ciclosporin A consistent with a substantial impact on cellularity in this key lymphoid organ.

19. Suppressed BCR-promoted Ca2+ responses, consistent with an inhibition of BCR-mediated signalling known to be critical for B cell development, self-tolerance (including structural or functional elimination of autoreactive B cells), and whose dysregulation is implicated across a breadth of B cell and pathogenic immunoglobulin-driven autoimmune disorders, including those with a T cell component.

20. Inhibited B cell activation in response to both T cell dependent and BCR-independent T cell- independent stimulation, a breadth of action expected to be of therapeutic relevance in

autoimmune disorders where either or both of these mechanisms of B cell activation initiate and/or perpetuate disease. Specifically, both clozapine and norclozapine have specific ability to inhibit anti- lg/CD40-L/IL-21- and CpG/IL-15-driven B cell activation and associated cytokine release.

21. Both clozapine and norclozapine are able to inhibit proliferation of B cells in response to CpG- ODN/IL-15 stimulation, of potential significance given the role self-containing immune complexes have in driving a TLR-dependent immune response.

22. Clozapine and norclozapine exert dose-dependent, functional inhibitory effects on B cell production of key cytokines implicated in the germinal centre response and/or inflammation: IL-6 and TNF-a. The ability of clozapine to suppress B cell-derived IL-6 secretion in response to multiple B cell stimuli may be of particular importance given the known key role of IL-6 produced by B cells in promoting the maturation of Tfh cells and its ability to induce spontaneous autoimmune germinal centre formation. These actions in vivo are fully congruent with the observed impact of clozapine to profoundly suppress primary and memory responses in the TDAR assay and the phenotype of Tfh cells in the collagen-induced arthritis model study.

23. Clozapine and norclozapine's impact on fundamental B cell biochemistry/signalling in vitro is seen to be comparable or, in some instances, even greater than that of highly potent established/ emerging classes of B cell-directed therapeutics targeting core proximal components of the BCR signal transduction machinery, pointing to a substantial B cell relevant pharmacology of clozapine and norclozapine expected to have high therapeutic potential in a range of disorders characterised by pathogenic autoreactive B cells, dysregulated B-T cell interaction (both germinal centre and extra- follicular) and pathogenic immunoglobulin and/.or B cell-derived cytokine production.

Thus, clozapine and norclozapine appear to have profound influence in vivo on the pathways involved in B cell maturation and pathogenic antibody (particularly pathogenic IgG and IgA antibody) production particularly via an impact on germinal centre T cell-B cell interaction, functionality and key regulators, likely potentiated by a reciprocal potentiation of immunosuppressive Foxp3 ⁺ Treg function. Clozapine is useful in treating pathogenic immunoglobulin driven B cell mediated diseases and treating pathogenic immunoglobulin driven B cell mediated diseases with a T cell component. Further, these findings are of substantial relevance to the process of IgE production by differentiated B cells based on the ontogeny of lgE ⁺ B cells and the production of IgE given that: IgE memory B cells and IgE plasma cells also develop via a germinal centre pathway (Talay et al., 2012); IgE switched memory B cells are the main source of cellular IgE memory (Talay et al., 2012); the ontogeny of lgE ⁺

B cells and plasma cells follows similar phenotypic stages to that for IgG(l), including lgE ⁺ germinal centre-like B cells, lgE ⁺ plasmablasts and lgE ⁺ plasma cells occurring via a sequential switching process from IgG (Ramadani et al., 2017); the intrinsic maturation state of B cells determines their capacity to undergo class switching to IgE with the highest proportion of lgE ⁺ cells derive from germinal centre B cells (Ramadani et al., 2017); isotype switching depends on the number of cell divisions and is greater for IgE than IgG (Tangye et al., 2002), consistent with the fact that IgE responses generally require more prolonged antigenic stimulation (Flasbold et al., 1998).

Accordingly, clozapine is expected to be useful in treating pathogenic immunoglobulin E (IgE) driven B cell mediated diseases.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

All patents and patent applications referred to herein are incorporated by reference in their entirety.

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