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`
`REVIEW
`
`International Immunology
`International Immunology, Vol. 27, No. 1, pp. 21–29
`doi:10.1093/intimm/dxu081
`doi:10.1093/intimm/dxu081
`Advance Access publication 20 August 2014
`
`© The Japanese Society for Immunology. 2014. All rights reserved.
`For permissions, please e-mail: journals.permissions@oup.com
`
`Therapeutic uses of anti-interleukin-6 receptor antibody
`
`Sujin Kang1,2, Toshio Tanaka1,2 and Tadamitsu Kishimoto3
`
`1Department of Clinical Application of Biologics, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita City,
`Osaka 565-0871, Japan
`2Department of Immunopathology, World Premier International Immunology Frontier Research Center, Osaka University, 3-1
`Yamada-oka, Suita City, Osaka 565-0871, Japan
`3Laboratory of Immune Regulation, World Premier International Immunology Frontier Research Center, Osaka University, 3-1
`Yamada-oka, Suita City, Osaka 565-0871, Japan
`
`Correspondence to: T. Kishimoto; E-mail: kishimoto@ifrec.osaka-u.ac.jp
`
`Received 13 July 2014, accepted 8 August 2014
`
`Abstract
`
`Cytokine-targeted therapy has generated a paradigm shift in the treatment of several immune-
`mediated diseases. Interleukin-6 (IL-6), which was initially identified as B-cell stimulatory factor 2, is
`a prototypical cytokine with wide-ranging biological effects on immune cells such as B and T cells,
`on hepatocytes, hematopoietic cells, vascular endothelial cells and on many others. IL-6 is thus
`crucially involved in the regulation of immune responses, hematopoiesis and inflammation. When
`infections and tissue injuries occur, IL-6 is promptly synthesized and performs a protective role
`in host defense against such stresses and traumas. However, excessive production of IL-6 during
`this emergent process induces potentially fatal complications, including systemic inflammatory
`response syndrome (SIRS), and dysregulated, persistently high expression of IL-6 causes the onset
`or development of various chronic immune-mediated disorders. For these reasons, IL-6 blockade
`was expected to become a novel therapeutic strategy for various diseases characterized by IL-6
`overproduction. Indeed, worldwide clinical trials of tocilizumab, a humanized anti-IL-6 receptor
`monoclonal antibody, have successfully proved its outstanding efficacy against rheumatoid arthritis,
`juvenile idiopathic arthritis and Castleman disease, leading to the approval of tocilizumab for
`the treatment of these diseases. Moreover, various reports regarding off-label use of tocilizumab
`strongly suggest that it will be widely applicable for acute, severe complications such as SIRS and
`cytokine-release syndrome and other refractory chronic immune-mediated diseases.
`
`Keywords: autoimmune diseases, chronic inflammatory diseases, IL-6, tocilizumab
`
`Introduction
`
`Cytokines are soluble regulators that facilitate intercellular com-
`munication in immune responses and hematopoiesis. Different
`from hormones, the characteristic features of cytokines are
`functional pleiotropy and redundancy (1). Although cytokines
`perform critical roles in host defense and maintenance of tis-
`sue homeostasis, abnormal production of cytokines causes
`the onset or development of acute and chronic diseases, so
`that novel therapeutic strategies using cytokines themselves
`or cytokine-targeted biologics have been developed and suc-
`cessfully used for the treatment of various diseases.
`Interleukin-6 (IL-6), composed of 184 amino acids, was
`originally identified as B-cell stimulatory factor 2 (BSF-2)
`that promotes immunoglobulin synthesis by activated B
`cells (2); its complementary DNA was successfully cloned in
`1986 (3). Later, IL-6 was found to be a prototypical cytokine
`with pleiotropic biological effects on immune responses,
`acute-phase responses and hematopoiesis, although some
`
`of these effects have been shown to be made redundant by
`those of other members of the IL-6 family of cytokines (4–6).
`IL-6 is not expressed in healthy individuals, but when
`infections or tissue injuries occur, IL-6 is rapidly synthesized
`and contributes to host defense (7). However, excessive
`production of IL-6 during this process has been implicated
`in the development of acute, severe complications, includ-
`ing systemic inflammatory response syndrome (SIRS) and
`cytokine-release syndrome (CRS), and chronic dysregu-
`lated production of IL-6 plays a pathological role in the onset
`and development of chronic immune-mediated diseases (5,
`8). For these reasons, it was anticipated that IL-6 blockade
`would constitute a novel therapeutic strategy for such dis-
`eases, resulting in the development of tocilizumab, a human-
`ized anti-IL-6 receptor antibody (8–10). This review article
`focuses on recent findings of IL-6-related research and pro-
`gress in IL-6-targeting therapy.
`
`Lassen - Exhibit 1021, p. 1
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`22 Therapeutic uses of anti-IL-6R, tocilizumab
`
`IL-6, a pleiotropic cytokine
`
`IL-6 exerts various biological effects on different target
`cells and organs (Fig.  1). When acting on hepatocytes, IL-6
`induces the synthesis of a wide range of acute-phase proteins,
`including C-reactive protein (CRP), serum amyloid A  (SAA),
`fibrinogen, hepcidin and α1-antichymotrypsin, but inhibits
`expression of fibronectin, albumin and transferrin (11, 12). At
`the site of infection or injury, IL-6 is rapidly generated from
`innate immune cells, including monocytes, macrophages and
`dendritic cells, via molecules that recognize pathogen-asso-
`ciated molecular patterns or damage-associated molecular
`patterns, thus triggering innate immune responses.
`IL-6 is also important for adaptive immune responses by pro-
`moting B- and T-cell differentiation. As originally reported, IL-6
`helps activated B cells to differentiate into antibody-producing
`cells, and it also promotes the growth of myelomas/plasmacyto-
`mas and enhances the survival of plasmablasts (13, 14). As one
`of its most important functions, IL-6 regulates the differentiation
`of naive CD4+ helper T cells into effector subsets. IL-6, together
`with TGF-β, preferentially induces differentiation of the naive
`CD4+ helper T cells into Th17 cells, which produce the inflamma-
`tory cytokine IL-17, but IL-6 inhibits TGF-β-induced differentia-
`tion of those cells into Treg cells. This results in an immunological
`imbalance between Treg/Th17 subsets, which is believed to be
`an important pathological mechanism for the development of
`autoimmune and chronic inflammatory diseases (15).
`
`Other actions of IL-6 are the promotion of T-follicular helper
`cell differentiation and IL-21 production (16), as well as induc-
`tion of cytotoxic CD8+ T-cell differentiation by augmenting
`expression of IL-2 and its receptor. During inflammation, IL-6
`increases the level of adhesion molecules and molecules that
`regulate migration, such as monocyte chemoattractant pro-
`tein-1 in endothelial cells (17). Finally, during hematopoiesis,
`IL-6, together with IL-3, synergistically affects the formation of
`pleiotropic blast cell precursors, which support the formation
`of macrophage and megakaryocyte differentiation.
`Besides its crucial involvement in the immune system, IL-6
`is also involved in bone homeostasis. When released from
`bone marrow stromal cells, it is required for the expression
`of the receptor activator of the NF-κB ligand (RANKL), and
`subsequently promotes osteoclast differentiation, through
`which systemic as well as peri-articular osteoporosis and
`joint destruction is induced, as seen, for example, in patients
`with rheumatoid arthritis (RA) (18). In fact, it has been found
`that an increase in serum IL-6 levels strongly correlates with
`severity of radiographically detectable joint destruction (19).
`Robust angiogenesis and vascular permeability are char-
`acteristic features of RA synovial tissues and these charac-
`teristics are mediated by excessive production of vascular
`endothelial growth factor (VEGF), whose synthesis is also
`up-regulated by IL-6 (20). In dermal tissue, IL-6 appears to
`promote keratinocyte proliferation and collagen production
`in dermal fibroblasts. Finally, IL-6 has been demonstrated to
`
`Fig. 1. Pleiotropic activity of IL-6 and the pathological implications in disease. IL-6 exerts a variety of biological effects by acting on various
`cells, and overexpression of IL-6 is pathologically involved in the status of various diseases, including immunological disorders, bone and car-
`tilage destruction, persistent acute-phase responses and angiogenesis.
`
`Lassen - Exhibit 1021, p. 2
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`interact with many other cells and organ systems, including
`vascular endothelial cells, the neuropsychological system
`and the hypothalamic–pituitary–adrenal endocrinal system.
`
`The IL-6 signaling system: IL-6 receptor and gp130
`
`IL-6 is structurally classified as a member of the four-helix-
`bundle family of cytokines (21) and interacts with two differ-
`ent types of molecules, namely, IL-6 receptor (IL-6R, CD126)
`(22) and the signal-transducing receptor subunit gp130
`(23). IL-6R exists in two forms, an 80-kDa transmembrane
`form and a 50–55-kDa soluble form. Transmembrane IL-6R,
`with a short cytoplasmic domain, interacts with gp130 and
`transduces the signal upon binding of IL-6, which is known
`as the classic IL-6 signaling pathway (Fig.  2). Due to the
`limited expression of transmembrane IL-6R on hepatocytes,
`monocytes, macrophages and lymphocytes, cell activation
`through the classic pathway is limited (24).
`Soluble IL-6R, which lacks a cytoplasmic region, can also
`form a complex with IL-6, leading to homodimerization of gp130
`and subsequent triggering of the downstream signaling cas-
`cade, which is known as the trans-signaling pathway. Soluble
`IL-6R is found in serum and tissue fluids, and gp130 is ubiq-
`uitously expressed in various cells, so that this trans-signaling
`
`Therapeutic uses of anti-IL-6R, tocilizumab 23
`
`pathway is widely used, and the pleiotropic activity of IL-6 can
`be explained by the broad range expression of gp130 (6, 25).
`gp130 is also used as a common signal-transducing molecule
`for the IL-6 family cytokines, including leukemia inhibitory
`factor, oncostatin M, ciliary neurotrophic factor, IL-11, cardio-
`trophin 1, cardiotrophin-like cytokine, IL-27 and IL-35. These
`mechanisms account for the redundancy of characteristic fea-
`tures of cytokines at the molecular level (26, 27).
`As schematically shown in Fig. 2, IL-6 stimulation causes
`tyrosine phosphorylation of a cytoplasmic protein, the
`kinase JAK that is constitutively bound with gp130, and
`then activates two main signal transduction pathways, the
`STAT3 pathway and the MAPK pathway, through phospho-
`rylation of the SH2-domain containing protein tyrosine phos-
`phatase-2 (SHP-2). STAT3 activated by JAK forms a dimer,
`translocates into the nucleus, and then acts as a transcrip-
`tion factor to induce expression of IL-6 responsive genes.
`Furthermore, STAT3 activated by IL-6–IL-6R interaction also
`induces SOCS1 and SOCS3 expression (28, 29). SOCS1
`binds directly to JAK, which attenuates its catalytic activ-
`ity. SOCS3, on the other hand, inhibits the signaling through
`directly binding to gp130. Putting these findings together,
`IL-6 signaling is controlled by SOCSs as negative feedback
`regulators.
`
`Fig. 2. The classic IL-6 signaling system. IL-6 is capable of activating two major pathways: the STAT3 and MAPK pathways. These pathways
`activate downstream signaling of IL-6R, leading to induction of a variety of gene expression. This signaling also induces SOCS1 and SOCS3
`expression, which in turn suppress the IL-6 signaling pathway.
`
`Lassen - Exhibit 1021, p. 3
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`24 Therapeutic uses of anti-IL-6R, tocilizumab
`
`Regulation of IL-6 production
`
`When infections or tissue damage occur, IL-6 synthesis is
`promptly induced to contribute to host defense. After removal
`of such stresses from the host, the IL-6 production ends as the
`result of strict control of IL-6 synthesis at the transcriptional
`and post-transcriptional stages. A number of cis-transcription
`factors have been identified to be involved in IL-6 gene acti-
`vation, including NF-κB, specificity protein 1 (SP1), nuclear
`factor IL-6 (NF-IL6), activator protein 1 (AP-1) and interferon
`regulatory factor 1 (IRF-1) (Table 1). Stimulation with TNF-α,
`IL-1, and even IL-6 activates the functional cis-regulatory fac-
`tors to bind to their specific elements at the 5′-flanking region
`in the IL-6 gene. IL-6 in liver in particular can strongly induce
`NF-IL6 activation, suggesting that NF-IL6 might be a main
`transcriptional factor for acute-phase protein expression. In
`addition to inflammatory cytokines, some virus products such
`as the human T-lymphotropic virus 1-derived transactivator
`protein (Tax) have been shown to modulate the DNA-binding
`activity of NF-κB and NF-IL6 to the IL-6 promoter region.
`On the other hand, inflammatory stimuli also modulate the
`expression of factors involved in IL-6 transcription suppres-
`sion, including the aryl hydrocarbon receptor (Ahr) (Table 1).
`Ahr is a ligand-activated transcription factor, which acts as
`a receptor for several exogenous toxins. It forms a complex
`with NF-κB and STAT1, leading to inhibition of the promoter
`activity of IL-6 in macrophages, so that Ahr-deficient mac-
`rophages feature enhanced production of IL-6 (30). In addi-
`tion, a number of studies have recently demonstrated that Ahr
`activation may also act on various immune cells such as T
`cells, B cells and dendritic cells, and thus affect their func-
`tion in immune responses (31–34). For example, the specific
`depletion of Ahr in T cells inhibits Th17 cell differentiation and
`the development of collagen-induced arthritis (33), and Ahr-
`deficient dendritic cells produce less of the anti-inflammatory
`cytokine, IL-10 (34). In addition, some micro-RNAs (miRs)
`reportedly modulate, either directly or indirectly, the bind-
`ing activities of several transcription factors to the IL-6 gene.
`Most post-transcriptional control mechanisms of cytokines
`target the 5′-untranslated region (UTR) or 3′-UTR of mRNAs
`to modify initiation of translation or stability, respectively
`(35). Modification of post-transcription of IL-6 mRNA primar-
`ily occurs at AU-rich elements located in the 3′-UTR region,
`and a number of RNA-binding proteins or miRs are involved
`in the regulation of IL-6 mRNA stabilization/degradation
`(Table  1). Interestingly, some recent studies have identified
`
`two counteractive molecules that regulate IL-6 mRNA stabil-
`ity (Fig.  3): regulatory RNase-1 (Regnase-1, also known as
`Zc3h12a) (36) and AT-rich interactive domain-containing pro-
`tein 5a (Arid5a) (37).
`Regnase-1 is a nuclease and binds to the 3′-UTR of IL-6
`mRNA, resulting in the destabilization of this mRNA. This
`resulted in the development of spontaneous autoimmune dis-
`eases, accompanied by splenomegaly and lymphadenopathy,
`in Regnase-1-deficient mice (36). Stimulation with an IL-1R/
`TLR agonist induces phosphorylation of Regnase-1, while the
`inhibitor of the NF-κB (IκB) kinase (IKK) complex destabilizes
`IL-6 mRNA. Phosphorylated Regnase-1 is subject to both
`ubiquitination and degradation. Regnase-1 also binds to the
`stem–loop site of the 3′-UTR of Regnase-1 mRNA and func-
`tions itself as a negative regulator. These findings demonstrate
`that IKK induces not only phosphorylation of IκB but also that
`of Regnase-1 to prevent IL-6 mRNA expression (38).
`We recently identified a novel RNA-binding protein, Arid5a
`(37) and found that its expression is rapidly induced and
`degraded within 6 h in macrophages upon LPS, IL-1β or
`IL-6 stimulation. Arid5a binds to the 3′-UTR of IL-6 mRNA
`and selectively stabilizes IL-6 but not TNF-α or IL-12 mRNA.
`Arid5a-deficient mice show a striking reduction in serum IL-6
`levels, induced by LPS injection, and in Th17 cell develop-
`ment in a mouse model—experimental autoimmune enceph-
`alomyelitis (EAE). Interestingly, Arid5a can interfere with the
`destabilizing effect of Regnase-1 on IL-6 mRNA, thus demon-
`strating that the balance between Arid5a and Regnase-1 is
`canonical for the stability of IL-6 mRNA, and also suggesting
`that predominance of Arid5a over Regnase-1 may give rise
`to the development of autoimmune inflammatory diseases
`(Fig.  3) (39). A  deeper understanding of the role of these
`RNA-binding proteins may thus result in novel therapeutic
`approaches to target IL-6 mRNA stability in various diseases.
`
`Pathological involvement of IL-6 in diseases
`
`The immediate and transient expression of IL-6 contributes to
`host defense against environmental stress factors. When the
`source of stress is removed from the host, the production of
`IL-6 is terminated and results in normalization of serum lev-
`els of acute-phase proteins such as CRP and SAA. However,
`IL-6 is also involved in disease development (4, 5). First,
`during immune response against infectious agents, exces-
`sive production of IL-6 induces potentially fatal complica-
`tions, such as SIRS and CRS. Second, chronic, dysregulated
`
`Table 1. Transcriptional and post-transcriptional regulation of IL-6 gene expression
`
`Function
`
`Protein
`
`Transcription
`
`Promotion
`
`Repression
`
`NF-κB, SP1, NF-IL6, AP-1, IRF-1, Tax, TAT, HBVX,
`mutant p53
`GR, ER, p53, PPARα, Ahr
`
`Post-transcription
`
`Stabilization
`Degradation
`
`P38α, ORF57, Arid5a
`TTP, BRF-1, BRF-2, Regnase-1
`
`microRNA
`
`—
`
`miR-155 (targeting NF-IL6), miR-146a/b
`(targeting IRAK1), miR-223 (targeting STAT3)
`—
`miR-365, miR-608
`
`Transcription of IL-6 is regulated by transcription factors or by miRs, and several RNA-binding proteins or miRs are post-transcriptional regula-
`tors for IL-6 mRNA. BRF, butyrate response factor; ER, estrogen receptor; GR, glucocorticoid receptor; HBVX, hepatitis B virus X protein; IRAK1,
`IL-1 receptor-associated kinase 1; ORF, open reading frame; PPARα, peroxisome proliferator-activated receptor α; Rb, retinoblastoma; TAT,
`human immunodeficiency virus 1-derived transactivator of the transcription protein; TTP, tristetraprolin.
`
`Lassen - Exhibit 1021, p. 4
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`Therapeutic uses of anti-IL-6R, tocilizumab 25
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`Fig. 3. Regulatory mechanisms of IL-6 production. Regnase-1 promotes degradation of IL-6 mRNA, whereas Arid5a counteracts this by desta-
`bilizing the effect of Regnase-1. The functional balance between Arid5a and Regnase-1 determines IL-6 mRNA stability. IRAK1, IL-1 receptor-
`associated kinase 1; NEMO, NF-κB essential modulator; TRAF6, TNF receptor-associated factor 6; Ub, ubiquitination.
`
`IL-6 production by particular cell populations leads to the
`development of various chronic immune-mediated diseases.
`However, at present the reason(s) for excessive or chronic
`IL-6 production remains unknown; it may be partly due to an
`imbalance between Arid5a and Regnase-1.
`The first evidence that IL-6 was pathologically involved
`in disease development was observed in a case of cardiac
`myxoma (40). The culture fluid obtained from the myxoma tis-
`sue of a patient with unclassified connective tissue disease,
`who had presented with fever, polyarthritis, increased serum
`CRP level, anemia and hypergammaglobulinemia with posi-
`tivity for anti-nuclear factor, contained a large quantity of IL-6
`and the myxoma tissues could be positively stained with anti-
`IL-6 antibody. Subsequent studies have demonstrated that
`excessive expression of IL-6 is found in synovial cells of RA,
`germinal center B cells in swollen lymph nodes of Castleman
`disease, myeloma cells and peripheral blood cells or infiltrat-
`ing cells in tissues involved in various other diseases, as well
`as in many tumor cells (4, 5, 8, 13).
`Moreover, the concept of the pathological role of IL-6 in dis-
`ease development has been supported by numerous experi-
`mental findings that IL-6 blockade by means of gene knockout
`or injection of neutralizing anti-IL-6 or anti-IL-6R antibody could
`
`prevent the onset or ameliorate the severity of diseases in ani-
`mal models. For instance, IL-6 blockade resulted in a striking
`reduction in susceptibility to Castleman disease-like symptoms
`in IL-6 transgenic mice, and also suppressed disease devel-
`opment in models of RA, systemic lupus erythematosus, sys-
`temic sclerosis, polymyositis, EAE, experimental autoimmune
`uveoretinitis and other autoimmune inflammatory diseases.
`On the basis of these findings, IL-6 targeting was expected to
`constitute a novel therapeutic strategy against immune-mediated
`diseases (8–10). This led to the development of tocilizumab, a
`humanized monoclonal antibody, with the CDR of a mouse anti-
`IL-6R grafted on to human IgG1 molecule (41). Tocilizumab can
`block both classic and trans-signaling pathways by inhibiting IL-6
`binding to transmembrane IL-6R and soluble IL-6R. The produc-
`tion of CRP in hepatocytes is mediated by the classic signaling
`pathway, and if the free concentration of tocilizumab is maintained
`in serum at more than 1 μg ml−1, CRP remains negative (42).
`
`Therapeutic uses of the anti-IL-6R antibody,
`tocilizumab
`
`The first clinical trial of tocilizumab was performed with seven
`patients with Castleman disease, a chronic lymphoproliferative
`
`Lassen - Exhibit 1021, p. 5
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`26 Therapeutic uses of anti-IL-6R, tocilizumab
`
`disorder characterized by multiple lymph node swellings with
`massive infiltration of mature plasma cells and persistent pro-
`duction of IL-6 in germinal center B cells. These patients had
`presented with severe inflammatory symptoms and labora-
`tory findings such as high fever, anemia, increased levels of
`acute-phase proteins, hypoalbuminemia and hypergamma-
`globulinemia, but the administration of tocilizumab promptly
`ameliorated clinical symptoms together with normalization of
`serum CRP levels and improvement of anemia, serum albu-
`min concentration and hypergammaglobulinemia (43). The
`outstanding efficacy of tocilizumab was subsequently con-
`firmed in another clinical trial with an enrollment of 28 patients
`with Castleman disease (44), and this resulted in the approval
`of tocilizumab as an orphan drug in 2005 in Japan.
`The first randomized controlled trial of tocilizumab for RA
`was performed in 45 patients, who were sequentially allo-
`cated to receive a single intravenous dose of either 0.1, 1, 5
`or 10 mg kg−1 of tocilizumab or placebo (45). At week 2, a sig-
`nificant difference was observed between the group treated
`with 5 mg kg−1 of tocilizumab and the placebo group, with
`five patients (56%) in the tocilizumab cohort and none in the
`placebo cohort achieving a 20% improvement in the defined
`range of symptoms formulated by the American College of
`Rheumatology (ACR20%).
`A 12-week, multicenter, double-blind, placebo-controlled
`late phase II trial was performed in Japan (46). In this trial,
`164 patients with refractory RA were randomized to receive
`either 8 or 4 mg kg−1 of tocilizumab every 4 weeks or placebo.
`At week 12, an ACR20% response was observed in 78, 57
`and 11% of RA patients treated with 8 mg kg−1 of tocilizumab,
`4 mg kg−1 of tocilizumab and placebo, respectively, while
`40% of patients in the 8 mg kg−1 group and 1.9% in the pla-
`cebo group achieved an ACR50% response. Subsequently,
`seven phase III clinical trials verified the outstanding efficacy
`of tocilizumab in the suppression of disease activity and pro-
`gression of joint destruction associated with RA, and this
`drug is currently approved for the treatment of RA in more
`than 130 countries (47).
`The European League Against Rheumatism now recom-
`mends tocilizumab as one of eight first-line biologics to be
`used for RA patients with an inadequate response to the
`standard disease-modifying antirheumatic drug (DMARD),
`methotrexate (MTX). First-line biologics include five TNF
`inhibitors (infliximab, adalimumab, golimumab, certolizumab
`and etanercept), a T-cell activation blocker (abatacept) and a
`B-cell depletory (rituximab) as well as tocilizumab. However,
`tocilizumab is the only biologic that has proved to be more
`efficacious as monotherapy than MTX or other DMARDs. TNF
`inhibitors require the concomitant use of MTX to achieve their
`maximal effects, but tocilizumab monotherapy is not inferior
`to the combination therapy of tocilizumab plus MTX in the
`suppression of disease activity (48).
`Since it is observed that MTX treatment reduces the plasma
`level of IL-6 but not that of TNF-α in patients with early RA
`(49), it is likely that the effect of MTX in RA is partly medi-
`ated via inhibition of IL-6 production and this may be one
`reason why tocilizumab does not require concomitant use of
`MTX for the maximal effect. Moreover, a direct comparison of
`tocilizumab and adalimumab, a fully human anti-TNF-α anti-
`body, demonstrated that as monotherapy tocilizumab was
`
`superior to adalimumab, as evaluated by several indices of
`disease activity in RA patients (50). For instance, at week 24,
`the proportion of patients attaining remission assessed by
`DAS28 (disease activity score in 28 joints) was 39.9% with
`tocilizumab and 10.5% with adalimumab. ACR20%, ACR50%
`and ACR70% response rates were achieved in 65% and
`49.4%, 47.2% and 27.8% and 32.5% and 17.9% of patients
`treated with tocilizumab and adalimumab, respectively. Thus,
`tocilizumab appears to be the most powerful antirheumatic
`biologic.
`In addition to the outstanding clinical efficacy, tocilizumab
`has unique properties in comparison with other biologics.
`First, as described elsewhere, IL-6 promotes the synthesis of
`acute-phase proteins such as SAA and hepcidin, which are
`proteins responsible for the development of amyloid A amy-
`loidosis and anemia of chronic disorder, respectively. Indeed,
`tocilizumab appears to be the most powerful suppressant to
`reduce the expression of these acute-phase proteins and the
`treatment reportedly produces prominent ameliorative effects
`on these complications (51, 52). Second, the predominance
`of Th17 cells over Treg cells in the effector CD4+ T-cell subsets,
`which is thought to be a fundamental immunological abnor-
`mality in RA, is possibly induced by continual expression of
`IL-6 (15); tocilizumab may correct this Th17/Treg imbalance.
`The results of recent studies have demonstrated that inhibi-
`tion of IL-6 function by tocilizumab could rectify the imbal-
`ance between Th17 and Treg cells in the peripheral CD4+ T-cell
`population (53–55).
`The third disease for which tocilizumab is currently on
`label is systemic juvenile idiopathic arthritis (sJIA), which is
`a subtype of chronic childhood arthritis that leads to joint
`destruction, functional disability and growth impairment,
`and is accompanied by systemic inflammation. A  clinical
`trial, composed of a 6-week open-label lead-in phase and a
`12-week double-blind phase, was performed for 56 children
`with sJIA in Japan (56). At the end of the open-label phase,
`tocilizumab treatment (8 mg kg−1, every 2 weeks) resulted
`in ACR Pediatric 30, 50 and 70% responses for 91, 86 and
`68% of the patients, respectively. Forty-three patients contin-
`ued to the double-blind phase and 16 (80%) of 20 patients in
`the tocilizumab group could maintain an ACR Pediatric 30%
`response, compared with only 4 (17%) of 23 patients in the
`placebo group.
`A global phase III trial, in which 112 children with active
`sJIA were enrolled, has also proved that tocilizumab is highly
`efficacious for the suppression of disease activity of sJIA (57),
`leading to the acknowledgement that a new era has started
`in the treatment of this disease, which had long been consid-
`ered to be one of the most intractable juvenile diseases.
`Moreover, various case studies, series and pilot stud-
`ies of off-label use with tocilizumab have produced favora-
`ble results, indicating that tocilizumab may be used for the
`treatment of various chronic, intractable immune-mediated
`diseases (9, 10, 39). These include systemic and organ-spe-
`cific autoimmune diseases, chronic inflammatory diseases,
`including autoinflammatory syndromes, and other diseases
`such as atherosclerosis, type 2 diabetes mellitus, atopic der-
`matitis, sciatica and amyotrophic lateral sclerosis (Fig. 4). In
`particular, accumulated evidence provides strong indications
`that tocilizumab looks highly promising for the treatment of
`
`Lassen - Exhibit 1021, p. 6
`
`

`

`Therapeutic uses of anti-IL-6R, tocilizumab 27
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`Fig. 4. The widespread application of the IL-6 targeting strategy for various diseases. Tocilizumab is currently used for the treatment of RA,
`systemic and polyarticular JIA and Castleman disease. Numerous favorable results from off-label use with tocilizumab suggest that it will be
`widely applicable for the treatment of other intractable diseases. RS3PE, remitting seronegative symmetrical synovitis with pitting edema.
`
`systemic sclerosis, large-vessel vasculitis, adult-onset Still’s
`disease, amyloid A amyloidosis and polymyalgia rheumatica,
`for all of which clinical trials are in progress.
`Neuromyelitis optica (NMO) is a chronic inflammatory
`demyelinating disease of the central nervous system that pri-
`marily affects the spinal cord and optic nerves. Autoantibodies
`against the astrocyte water channel protein, aquaporin-4
`(AQP-4), play a pathological role in the disease development.
`Anti-AQP-4 antibodies are produced by the plasmablast
`population showing a CD19intermediateCD29highCD38highCD180−
`phenotype that is increased in the peripheral blood of NMO
`patients (58). IL-6 enhances the survival of the plasmablast
`population, whereas the addition of tocilizumab into the cul-
`ture diminishes the survival (58). These findings suggest that
`an IL-6-blockage strategy is promising for the treatment of
`NMO by inhibiting anti-AQP-4 antibody production. Indeed,
`the prominent beneficial effects of tocilizumab have been
`recently reported in patients with NMO that is refractory to
`conventional treatment regimens (59–61), and a clinical trial
`of a new fully human antibody against IL-6R (SA237) (62)—
`generated from tocilizumab by techniques for structural anti-
`body optimization—for NMO is in progress.
`In addition to these indications of the potential of tocili-
`zumab for the successful treatment of a variety of chronic
`
`diseases, an IL-6-blockade strategy could serve as rescue
`therapy for acute life-threatening conditions. CRS entails
`potentially fatal immediate complications and is sometimes
`induced by non-physiologic T-cell activation after therapies
`that engage T cells by using a chimeric, modified antigen-
`receptor or by using a CD19–CD3-bi-specific antibody
`(blinatumomab) (63). IL-6 and IL-10 as well as the effector
`cytokine, IFN-γ, have been shown to be markedly elevated
`in patients with CRS. Surprisingly, one administration of toci-
`lizumab for three patients with CRS receiving T-cell-engaging
`therapies dramatically resolved their serious conditions (64,
`65). These findings suggest that this IL-6R antibody may
`constitute a novel therapeutic drug for emergent fatal com-
`plications mediated by a cytokine storm, such as CRS, SIRS,
`macrophage activation syndrome, hemophagocytic syn-
`drome or septic shock.
`
`Conclusions
`
`Following the successful cloning of the IL-6 gene, IL-6-related
`research has progressed rapidly (8). Clarification of the whole
`picture of the IL-6-mediated signaling system solved the long-
`standing mystery of the functional pleiotropy and redundancy
`of cytokines. In line with this progress, the pathological role
`
`Lassen - Exhibit 1021, p. 7
`
`

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`28 Therapeutic uses of anti-IL-6R, tocilizumab
`
`of IL-6 in various diseases has been thoroughly documented,
`and this resulted in the development of the humanized anti-
`IL-6R monoclonal antibody, tocilizumab.
`Clinical trials of tocilizumab started in the late 1990s,
`and this biologic was first approved for the treatment of
`Castleman disease in Japan in 2005. During the following
`years, tocilizumab has been adopted as a first-line biologic
`for the treatment of RA and is currently being used in more
`