Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Review
`
`Interleukin-6 as a Therapeutic Target
`Jean-Fran¸cois Rossi1,2,3, Zhao-Yang Lu4, Michel Jourdan2, and Bernard Klein2,3
`
`Clinical
`Cancer
`Research
`
`Abstract
`
`Human IL6 is a cytokine produced by many cell types that
`has pleiotropic effects. In agreement, anti-IL6 therapy reduces
`inflammation, hepatic acute phase proteins, and anemia and
`has antiangiogenic effects. Blocking IL6 has demonstrated
`therapeutic efficacy with drug registration in Castleman disease
`and inflammatory diseases (rheumatoid arthritis) without
`major toxicity. Interestingly, the inhibition of C-reactive pro-
`tein (CRP) production is a trustworthy surrogate marker of anti-
`IL6 therapy efficacy. Clinically registered IL6 inhibitors include
`siltuximab, an anti-IL6 mAb, and tocilizumab, an anti-IL6R
`mAb. In various cancers, in particular plasma cell cancers, large
`randomized trials showed no efficacy of IL6 inhibitors, despite
`
`a full inhibition of CRP production in treated patients in vivo,
`the numerous data showing an involvement of IL6 in these
`diseases, and initial short-term treatments demonstrating a
`dramatic inhibition of cancer cell proliferation in vivo. A likely
`explanation is the plasticity of cancer cells, with the presence of
`various subclones, making the outgrowth of cancer subclones
`possible using growth factors other than IL6. In addition,
`current therapeutic strategies used in these cancers already
`target IL6 activity. Thus, anti-IL6 therapeutics are able to neu-
`tralize IL6 production in vivo and are safe and useful in inflam-
`matory diseases and Castleman disease. Clin Cancer Res; 21(6);
`1248–57. Ó2015 AACR.
`
`Introduction
`
`IL6 was first described as a cytokine inducing B lymphocytes
`to produce immunoglobulin or stimulating hepatocytes and it
`was named B-stimulating factor-2 or hepatocyte growth factor
`(HGF; ref. 1; Figs. 1 and 2). IL6 is an inflammatory cytokine
`involved in various biologic processes, including dysimmune
`diseases and cancers (2, 3). It
`is produced by many cell
`lineages, including stromal cells, hematopoietic cells, epithe-
`lial cells, or muscle cells. IL6 binds to a membrane receptor
`(IL6R or CD126) or to its soluble form (sIL6R) with a 109 Kd
`(4). Then, the complexes IL6/IL6R or IL6/sIL6R bind to the
`gp130 IL6 transducer (CD130) with a low Kd (1011 M). These
`bindings result in gp130 dimerization, phosphorylation, and
`activation of
`receptor-associated kinases
`(JAK1,
`JAK2,
`and Tyk2; ref. 4). Gp130 is a common transducing chain used
`by the seven members of the gp130 cytokine family and by
`IL27 (5).
`Anti-IL6 therapies have been developed for the treatment of
`dysimmune diseases and in cancers. Current IL6 inhibitors
`include mAbs to IL6 (siltuximab) or IL6 receptor (tocilizu-
`mab), and other inhibitors are being investigated in clinical
`trials. This review updates the data on the clinical efficacy of
`IL6 inhibitors.
`
`1Department of Hematology, CHU de Montpellier, Montpellier, France.
`2INSERM U1040, Montpellier, France. 3Universite Montpellier
`I,
`Montpellier, France. 4Unite de Therapie Cellulaire, CHU de Montpellier,
`Montpellier, France.
`
`Note: J.-F. Rossi and B. Klein share senior authorship.
`
`Corresponding Author: Jean-Fran¸cois Rossi, Department of Hematology, CHU
`Saint-Eloi, 80 Avenue Augustin Fliche 34295 Montpellier Cedex, France. Phone:
`33-4-67-33-80-79; Fax: 33-4-67-33-83-73; E-mail: jeanfrancoisrossi@me.com
`
`doi: 10.1158/1078-0432.CCR-14-2291
`
`Ó2015 American Association for Cancer Research.
`
`Lessons from Anti-IL6 Murine Monoclonal
`Antibodies
`
`In the early 1990s, our group initiated the first clinical trials
`with anti-IL6 murine mAbs in patients with multiple myeloma or
`metastatic renal cell carcinoma (MRCC).
`
`Clinical observations
`The first treated patients had extramedullary multiple myeloma
`disease, in particular plasma cell leukemia, and the inhibition of
`malignant plasma cell proliferation within a few days of treatment
`confirmed that IL6 was a growth factor for malignant plasma cells
`in vivo (6, 7), as shown using in vitro models (8, 9). These first anti-
`IL6 treatments used anti-IL6 murine mAbs, elsilimomab (BE-8),
`and/or BE-4, or mAb8, and have shown that the C-reactive protein
`(CRP) production by human hepatocytes was completely under
`the control of IL6 in vivo, as evidenced by the loss of circulating
`CRP throughout anti-IL6 treatment and a quick loss reversal at
`treatment discontinuation (7). Anti-IL6 therapy also inhibited
`fever with body temperature normalization (7) in line with IL6
`pyrogenic activity in rats (10). No major toxicity was evidenced,
`except a transient and mild (25%) decrease in platelet count
`(7, 11–13), which is expected given the role of IL6 to drive
`megakaryocyte maturation (14). Objective response with a
`decrease of the monoclonal component was observed in some
`patients, including refractory patients (11). We also treated 18
`patients with MRCC with BE-8 (elsilimomab) and 4 patients
`received both BE-8 and IFNa-2a, resulting in 2 partial responses,
`2 minor responses, one stable disease (12). Patients' conditions
`improved, in particular with an increase in hemoglobin level,
`analgesia, and a lack of flu-like syndrome despite IFN coadmin-
`istration in some patients (12). Serum CRP was no more detect-
`able within 2 days after start of anti-IL6 treatment, making it
`an easy marker of treatment efficacy (7, 11, 12). Other acute
`phase proteins also decreased rapidly (serum amyloid A protein,
`a1 anti-trypsin) and serum albumin increased 20 days after the
`anti-IL6 therapy initiation in agreement with the major role of
`IL6 to control acute phase protein and albumin production by
`
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`on July 19, 2019. © 2015 American Association for Cancer Research.
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`Lassen - Exhibit 1024, p. 1
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`

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`Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Anti-IL6 Therapy
`
`Inflammation Immunity Reproduction Metabolism Hematopoiesis
`
`Neural development Bone remodeling Angiogenesis
`
`Figure 1.
`IL6 is a pleiotropic cytokine. gp130,
`glycoprotein 130; Treg, regulatory
`T cell.
`
`IL6 (4-helix bundle, 212 AA)
`
`B lymphocytes: plasma cell differentiation,
`survival/proliferation factor for multiple
`myeloma
`
`T lymphocytes: in association with TGFβ
`differentiation of TH17 and IL27 inhibition
`of Treg
`
`Megakaryocytes: differentiation
`
`Cardiac stem cells: cell survival
`
`Adipocytes: lipolysis
`Osteoclasts: Cox-2, Wnt, NF-κB, RANK
`
`Neural stem cells: astrocyte differentiation
`
`© 2015 American Association for Cancer Research
`
`hepatocytes (15). Hemoglobin level slightly increased by 1 to 1.5
`g/dL due to inflammation control and no major change was seen
`in circulating T cells, natural killer cells, or B lymphocytes and
`in complement molecules. Overall, these first clinical trials with
`
`murine anti-IL6 mAb have shown the lack of toxicity of anti-IL6
`therapy, identified CRP as an easy and quick surrogate marker of
`IL6 bioactivity, and shown antitumor effects in some patients with
`multiple myeloma or MRCC. In addition, they made it possible to
`
`sIL6R
`
`IL6/sIL6R
`
`1
`
`IL11
`
`CLC
`
`CNTF
`
`LIF
`OSM
`
`CT-1
`
`OSM
`
`2
`
`IL27
`
`Free
`IL6
`
`gp130
`
`WSX-1
`
`PI3K
`
`AKT
`
`IL6R
`
`IL11R
`
`P P
`
`JAK
`
`STAT3
`
`STAT3
`
`STAT3
`
`P
`
`P
`
`MAPK
`
`Survival and cell cycle
`Increased RANKL expression
`
`Cell cycle
`Cell growth
`
`Survival
`
`© 2015 American Association for Cancer Research
`
`Figure 2.
`The IL6 receptor and glycoprotein
`(gp) 130 transducer cytokine family.
`Part 1 illustrates usual signaling. Part 2
`shows trans-signaling. AKT, protein
`kinase B; CLC, cardiotrophin-like
`cytokine; CNTF, ciliary neurotrophic
`factor; CT-1, cardiotrophin-1; gp,
`glycoprotein; LIF, leukemia inhibitory
`factor; OSM, oncostatin M; WSX-1, IL27
`receptor subunit a.
`
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`Clin Cancer Res; 21(6) March 15, 2015
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`on July 19, 2019. © 2015 American Association for Cancer Research.
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`1249
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`Lassen - Exhibit 1024, p. 2
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`

`

`Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Rossi et al.
`
`understand the main biologic mechanisms involved in anti-IL6
`therapy.
`
`Basis for Pharmacologic Effects of
`Antagonists
`
`Initial investigations of the biologic mechanisms involved in
`anti-IL6 treatment
`At the stop of the anti-IL6 mAb treatment, a quick recovery of
`CRP production, fever, and cancer disease was observed (7). The
`reason is that the anti-IL6 mAb binds IL6, prevents its consump-
`tion by cells and its renal clearance, and accumulates large levels of
`circulating IL6 in the form of stable monomeric IL6/anti-IL6
`complexes (16). The half-life of circulating IL6 increased from
`a few minutes in untreated individuals to several days, actually the
`same half-life of the free mAb, in anti-IL6–treated patients (16).
`Using this observation, we calculated the in vivo daily production
`of IL6 by integrating pharmacologic and affinities parameters and
`showed a high variation in daily IL6 production in vivo, from
`several mg/day to mg/day, and in patients with the lower IL6
`production, a complete blockade of CRP production and objec-
`tive response (17). In a patient developing acute Escherichia coli
`septicemia during anti-IL6 treatment, a production of IL6 over
`7 mg/day was calculated (18). Using CRP serum levels as a
`surrogate marker for IL6 bioactivity in vivo, we have shown that
`the anti-IL6 mAb was able to fully block IL6 activity in patients
`producing less than 18 mg IL6 per day, in association with an
`antitumor effect (11, 17). This finding indicated that the dose of
`anti-IL6 mAb injected could be several 100-fold too low to
`neutralize a huge IL6 production in vivo and to control CRP
`production and tumor growth in some patients.
`In addition, at the end of anti-IL6 mAb treatment, we have
`shown that the ratio of the concentrations of free anti-IL6 mAbs to
`IL6/anti-IL6 mAb complexes decreased, making it possible for
`soluble or cell membrane IL6 receptors to disrupt IL6/anti-IL6
`mAb complexes and use this large amount of circulating IL6 to
`trigger cell activation. This mechanism explains the quick recovery
`of CRP production, fever, and cancer cell progression occurring at
`the end of anti-IL6 treatment in some patients (11). A possibility
`to avoid this rebound effect is to use a combination of two or three
`anti-IL6 mAbs recognizing different epitopes. This would result in
`the formation of polymeric IL6/IL6 mAb complexes, which are
`captured mainly by hepatocytes leading to a rapid clearance, as we
`demonstrated in a murine model (19).
`
`The Anti-IL6 Drugs
`
`Several anti-IL6 mAbs have been developed. The CNTO328
`chimeric anti-IL6 mAb (siltuximab) was used in clinical trials for
`multiple myeloma (20, 21), MRCC (22), and prostate cancer (23)
`and was recently registered for treating patients with Castleman
`disease (24). Sirukumab, a humanized anti-IL6 mAb, has been
`assayed in healthy subjects to determine PK/PD and safety. This
`drug has a half-life ranging from 18.5 to 29.6 days with no serious
`adverse events (25). Anti-IL6R mAb, particularly atlizumab (also
`called tocilizumab), has been assayed in dysimmune diseases
`(26). Recently, CytomX Therapeutics Inc. developed targeted
`"masked" antibodies, which are activated by disease-associated
`proteases (27). Sant7, a potent antagonist of the IL6 receptor,
`was engineered through targeted amino acid substitutions in
`key residues of the human IL6 molecule (28). Sant7 shows
`higher affinity than IL6 for the gp80 receptor subunit, but
`completely lacks binding capacity to the gp130 receptor sig-
`naling subunit. Other inhibitors have been developed (Tables 1
`and 2).
`
`In the initial clinical trials with the BE-8 anti-IL6 murine mAb, a
`full inhibition of CRP production was achieved only in patients
`producing less than 18 mg/day of IL6 (11). Considering the
`molecular weights of IL6 and BE-8 anti-IL6 mAb, the Kd of
`binding of BE-8 to IL6 (1011 M), and the concentration of
`circulating BE-8 in patients (mean concentration of 10 mg/mL),
`a production of IL6 less than 18 mg/day in vivo meant a 100-fold
`molar excess of BE-8 mAb to IL6 (Fig. 3). In patients with multiple
`myeloma treated with the siltuximab humanized anti-IL6 mAb, a
`siltuximab concentration of 5 mg/mL was proposed to achieve a
`300-fold molar excess of siltuximab to IL6, which is considered
`necessary to block IL6 activity and tumor growth with that
`mAb (29). In a phase I study in patients with MRCC, the admin-
`istration of 6 mg/kg of siltuximab every 2 weeks efficiently
`suppressed serum CRP in patients who had a baseline CRP level
` 30 mg/L (30). These data fit well with the pharmacokinetics
`parameters of siltuximab (30). Indeed, clinically relevant sche-
`dules of siltuximab were simulated predicting a dosage of 6 mg/kg
`every 2 weeks or 9 mg/kg every 3 weeks would decrease CRP to
`below the lower limit of quantification (30).
`Regarding tocilizumab, an administration of 8 mg/kg once
`every 2 weeks resulted in a marked increase in serum sIL6R in
`the form of sIL6R/tocilizumab complexes and reached a steady
`state at day 42 of treatment (31). A decrease of CRP level was
`found as long as the concentration of free tocilizumab not
`engaged in sIL6R/tocilizumab complexes and able to inhibit
`biding of IL6 to membrane or soluble IL6R remained above
`1 mg/mL in serum (31).
`
`Diseases Improved by Anti-IL6 Therapies
`
`Dysimmune diseases
`In the early phases of inflammation, IL6 is produced by
`monocytes and macrophages, in particular though the stimu-
`lation of Toll-like receptors. A deregulated and persistent IL6
`production has been observed in various chronic inflammatory
`and/or autoimmune diseases, including in animal models. IL6
`blockade by means of gene-knockout or administration of anti-
`IL6 or anti-IL6R antibody can suppress such disease develop-
`ment either preventively or therapeutically. The anti-IL6R mAb
`tocilizumab has demonstrated efficacy either as a monotherapy
`or in combination with disease-modifying antirheumatic drugs
`for adult patients with moderate to severe rheumatoid arthritis
`(for review refs. 26, 31). A Cochrane database systematic review
`concluded that tocilizumab-treated patients were four times
`more likely to achieve American College of Rheumatology 50%
`improvement (38.8% vs. 9.6%) and 11 times more likely to
`achieve Disease Activity Score remission as compared with
`control patients (30.5% vs. 2.7%; ref. 32). Thus, the anti-IL6R
`mAb tocilizumab is now approved for the treatment of rheu-
`matoid arthritis in more than 90 countries. The outstanding
`results obtained with tocilizumab in rheumatoid arthritis led to
`a change in the treatment objective from protection against
`joint destruction to prolongation of life expectancy with nor-
`mal activities in daily life. Safety has been reported from six
`studies performed in Japan. The incidence of adverse events
`(AE), including abnormal laboratory test results, was 465.1 per
`100 patient-years, with infection being the most common AE
`
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`on July 19, 2019. © 2015 American Association for Cancer Research.
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`Clinical Cancer Research
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`Lassen - Exhibit 1024, p. 3
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`

`

`Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Anti-IL6 Therapy
`
`Table 1. Anti-IL6 antagonists in clinical trials
`
`Drug (company)
`
`Elsilimomab or BE-8
`
`Siltuximab, chimeric (CNTO328)
`
`Sirukumab, CNTO 136
`Humanized anti-IL6
`ALD518 or BMS-945429
`
`Anti-IL6, humanized
`
`Alder Biopharm. Inc./BMS
`
`Clinical trial (disease/type)
`
`ClinicalTrials.gov
`
`Lymphoma, MM, MRCC
`Phase I/II
`Mab 1339, fully human from BE-8 OPi EUSA/Vaccinex/GSK
`MM
`Phase I
`Phase II multicentric CNTO 328 þ Dex
`Randomized phase II, multicentric CNTO328 þ Bor
`First-line phase Ib/II Len/Bor/Dex/CNTO328
`MGUS, SMM, indolent MM
`Phase I (effect on heart)
`SMM high-risk
`Phase II randomized
`Myelodysplastic syndrome
`Randomized phase II
`CNTO328þBSC vs. placeboþBSC
`Solid cancers
`Phase I/II, ovarian, pancreatic, colon, head and neck, lung cancers
`MRCC
`Phase I/II
`Prostate cancer metastatic, hormone-refractory
`Mitoxantrone þ prednisone þ CNTO328 phase II
`Prostate cancer not responding to hormone therapy
`Multicentric Castleman disease
`Randomized phase II (long-term evaluation)
`Active RA
`Under methotrexate therapy
`Cancer
`Phase I
`NSCLC
`Phase I/II (fatigue and cachexia) multicentric
`RA
`Phase II
`Crohn disease
`Phase II (discontinued)
`
`NCTO1309412
`NCT00402181
`
`NCTO1531998
`
`NCT01219010
`
`NCT01484275
`
`NCT015133317
`
`NCT00841191
`
`NCT00385827
`
`NCT00433446
`NCT0400503
`
`NCT00718718
`
`NCT00867516
`
`NCT01545050
`
`Olokizumab (CDP6038)
`Humanized anti-IL6
`UCB Group/Russia's R-Pharm
`
`RA
`Anti-TNF failure phase II
`
`NCT01533714 and NCT01463059
`
`Abbreviations: Bor, bortezomib; BSC, best supportive care; Carbo, carboplatin; Dex, dexamethasone; Doxo, doxorubicin; Len, lenalidomide; MGUS, monoclonal
`gammopathy of undetermined significance; MM, multiple myeloma; NSCLC, non–small cell lung carcinoma; RA, rheumatoid arthritis; SMM, smouldering multiple
`myeloma.
`
`with 6.22 per 100 patient-years (32). As expected, increases in
`liver function and lipid parameters were observed. Tocilizumab
`is also a promising drug for systemic lupus erythematosus,
`systemic sclerosis, polymyositis, Takayasu and giant cell arter-
`itis, Crohn disease, relapsing polychondritis, multiple sclerosis,
`Still disease, and Beh¸cet disease (26, 31).
`
`Castleman disease
`Castleman disease is a lymphoproliferative disease character-
`ized by benign hyperplastic lymph nodes, follicular hyperplasia
`with polyclonal plasmablastic proliferation and capillary prolif-
`eration associated with vascular hyperplasia and high IL6 activity,
`mainly due to viral IL6 (33, 34). Viral IL6 is produced by cells
`infected by Kaposi sarcoma-associated herpes virus (KSHV) and
`directly binds and stimulates gp130 IL6 transducer in the absence
`of IL6R (33, 34). All of the HIV-positive and half of the HIV-
`negative patients with multicentric Castleman disease were
`infected with KSHV (33). The rationale for developing anti-IL6
`therapy was first that IL6 is a main growth factor for plasmablasts
`(35) and that it has pleiotropic activities, which may explain
`vascular hyperplasia in particular. In particular, transgenic mice
`bearing an IL6 transgene driven by the immunoglobulin Em
`enhancer develop massive polyclonal plasmacytoses and rapidly
`die (36).
`
`The BE-8 anti-IL6 murine mAb was first used for a patient with
`Castleman disease, who showed obvious disease improvement
`after treatment (37). A clinical trial with the anti-IL6R mAb
`tocilizumab confirmed this benefit and led to its approval in
`Japan as an orphan drug for this disease in 2005. Recently, the
`anti-IL6 mAb siltuximab was also proven to provide major clinical
`benefit in a randomized phase II study enrolling 79 patients, and it
`has been approved for treatment of this disease (24).
`
`Diseases with No Demonstrated Benefit of
`Anti-IL6 Therapies
`
`Multiple myeloma
`Before reviewing the efficacy of randomized anti-IL6 trials in
`patients with multiple myeloma, it is worthwhile to update the
`current knowledge about the place of IL6 as a growth factor for
`multiple myeloma cells (MMC). IL6 was recognized in the late
`1980s as an important growth factor for MMCs, being produced
`mainly by the tumor environment (6, 7) and also by MMCs (8).
`Although most studies confirmed these findings (38), current
`data indicate that insulin-like growth factor-1 (IGF-1) is the
`major growth factor for MMCs, and that IL6 is active mainly in
`CD45þ MMCs (39, 40). The phosphatase CD45 dephosphor-
`ylates IGF-1R in MMCs and weakens IGF-1R signaling, making
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`Clin Cancer Res; 21(6) March 15, 2015
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`1251
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`Lassen - Exhibit 1024, p. 4
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`

`

`Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Rossi et al.
`
`Table 2. Anti-IL6 receptor antagonists in clinical trials
`
`Clinical trial (disease/type)
`
`208 studies including
`Ovarian cancer
`Phase I/II
`(Carbo/pegylated liposomal doxorubicin hydrochloride or Carbo/Doxo) þ
`tocilizumab/Peginterferon alfa-2b
`Erdheim–Chester disease
`(histiocytose)
`Phase II
`Hematophagocytic lymphohistiocytosis
`Phase II
`Graft-versus-host disease
`Phase II
`Steroid-refractory
`Castleman disease KSHV
` Antiviral drugs
`Continuing in responding patients
`14 studies: 12 in RA, including a phase III trial,
`1 in noninfectious uveitis, and 1 in ankylosing spondylitis
`
`ClinicalTrials.gov
`
`NCT01637532
`
`NCT01727206
`
`NCT02007239
`
`NCT02174263
`
`NCT01475162
`NCT01441063
`
`NCT01183598
`
`Drug (company)
`
`Tocilizumab
`
`Sarilumab IL6R
`Sanofi/Regeneron
`VX30 (Vaccinex)
`
`ARGX-109 (arGEN-X)
`
`FM101 (Formatech)
`
`Sant7 (receptor superantagonist)
`
`Abbreviations: Bor, bortezomib; BSC, best supportive care; Carbo, carboplatin; Dex, dexamethasone; Doxo, doxorubicin; Len, lenalidomide; MGUS, monoclonal
`gammopathy of undetermined significance; MM, multiple myeloma; NSCLC, non–small cell lung carcinoma; RA, rheumatoid artritis; SMM, smouldering multiple
`myeloma.
`
`MMCs more dependent on IL6 (39). Besides IGF-1 and IL6,
`other MMC growth factors have been described, including
`mainly BAFF/APRIL, produced by osteoclasts (41), and mem-
`bers of the EGF family, such as HGF and VEGF (see ref. 42 for
`review). In addition, CRP, whose production is under IL6
`control, also increases IL6 production in MMCs, enhances their
`
`proliferation under stress conditions, and protects them from
`chemotherapy (43).
`Regarding signaling pathways, IL6 triggered the JAK/STAT3
`pathway, which drives an antiapoptotic response in MMCs,
`mainly through upregulation of the MCL1 antiapoptotic protein
`)44 ). It also triggers the MAP kinase ERK1/2, activating the cell
`
`sIL6R
`
`10–11 M
`
`IL6
`
`10–9 M
`
`10–9 M
`
`10–11 M
`
`Anti-IL6
`mAb
`
`10–11 M
`
`Figure 3.
`Basis for pharmacologic effects of IL6
`antagonists. gp, glycoprotein.
`
`IL6R
`
`gp130
`
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`© 2015 American Association for Cancer Research
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`on July 19, 2019. © 2015 American Association for Cancer Research.
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`Clinical Cancer Research
`
`Lassen - Exhibit 1024, p. 5
`
`

`

`Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Anti-IL6 Therapy
`
`cycle (41). The PI3K and AKT signaling pathway is also important
`to promote MMC growth. The AKT kinase is activated in MMCs
`(45). But, in our opinion, IL6 does not activate this pathway,
`unlike IGF-1 or other MMC growth factors (44). This may explain
`the additive effects between IL6 and other growth factors to
`support MMC growth.
`Two randomized trials were recently published showing a
`lack of effect of anti-IL6 therapy. In one trial, the anti-IL6 mAb
`silxutimab was given to transplant-ineligible patients in asso-
`ciation with a regimen of bortezomib–melphalan–prednisone
`compared with bortezomib–melphalan–prednisone and then
`as a maintenance therapy for 18 months or until relapse (20).
`In another trial, siltuximab was given in relapsed/refractory
`multiple myeloma in association with bortezomib compared
`with bortezomib alone and then as maintenance therapy until
`progression (21). These two trials showed no benefit for event-
`free or overall survivals. In the first study, an improvement
`of response was found. The initial anti-IL6 treatments per-
`formed by our group did not investigate event or overall
`survival, but showed a quick blockade of tumor proliferation
`and reduction in tumor mass in patients with fulminant dis-
`ease, mainly with extramedullary proliferation (7, 11). In a
`series of 24 newly diagnosed patients, the BE-8 anti-IL6 mAb
`was given together with 140 mg/m2 of melphalan and autol-
`ogous stem cell transplantation for 21 days (13). This combi-
`nation was shown to be as active as 200 mg/m2 of melphalan to
`decrease the tumor mass, based on historical comparison, and
`without impairment on the hematopoietic recovery (13).
`How can we explain this lack of benefit in randomized trials
`with anti-IL6 mAb despite the major role of IL6 to control healthy
`plasmablast proliferation (35, 36) and despite the blockage of
`myeloma cell proliferation by anti-IL6 mAb in patients with
`fulminant disease (7, 11)? In these randomized trials, siltuximab
`was able to block CRP production, indicating its neutralization of
`IL6 bioactivity in the liver. Thus, a lack of antimyeloma effect of
`siltuximab was not due to an inability to block a too large IL6
`production. In line with this, median CRP levels are not higher in
`patients with multiple myeloma compared with those with rheu-
`matoid arthritis for whom anti-IL6 therapy had benefit (Table 3).
`Several mechanisms could explain this lack of effect of anti-IL6
`therapy. First of all, the efficacy of anti-IL6 therapy was compared
`with drug combination (bortezomib or bortezomib–melphalan–
`prednisone), which already reduces IL6 production and inhibits
`CRP production (20). Second, as summed up above, other factors
`could stimulate MMC growth in the absence of IL6, in particular
`IGF1 in CD45/low MMCs, and favor the emergence of MMC
`subclones independent of IL6 (39, 40). This emergence of IL6-
`independent subclones could not be investigated in our initial
`short-term treatments in patients with fulminant disease. Third,
`other cytokines of the IL6 family can trigger the gp130 IL6
`transducer and also the growth of MMCs and could substitute
`for IL6 to promote MMC survival and growth in vivo (46).
`
`Other hematologic malignancies
`IL6 and/or CRP are prognostic factors for several B-cell malig-
`nancies, particularly malignant lymphoma (47). IL6 is a biologic
`marker in other lymphoid malignancies, including Hodgkin
`disease, mantle-cell lymphoma, cutaneous CD30-positive lym-
`phomas, and KSHV-associated malignancies (48–52). Poly-
`morphisms of different cytokine genes, and particularly of IL6
`gene (IL6-174GG genotype) are associated with treatment failure
`
`in Hodgkin disease (53). The majority of B-cell tumors expressed
`the IL6 receptors, in particular mantle cell lymphoma (54). BE-8
`was used in 11 patients with HIV-associated lymphoma and
`showed a clinical benefit, particularly on B symptoms (55), and
`in one patient with acute monoblastic leukemia (56). Therefore,
`IL6 and more generally gp130 IL6 transducer signaling could play
`an important role in the pathogenesis of certain B-cell neoplasias,
`and new clinical programs could be developed in such diseases.
`
`Metastatic renal cell carcinoma
`IL6 was shown to be an autocrine proliferation factor for tumor
`cell lines obtained from patients with MRCC (57). Mutations in
`the TP53 gene could contribute to the overexpression of IL6 in
`renal cell carcinoma (58). We and others have demonstrated that
`CRP/IL6 is a prognostic factor in MRCC (12, 59, 60). Furthermore,
`we demonstrated a significant association between the presence of
`the IL6R in tumors and tumor stage, nuclear grade, proliferation
`index, and serum IL6 (61). Treatment of renal cell carcinoma cells
`with cisplatinum (CDDP) in combination with anti-IL6 or anti-
`IL6R mAbs can overcome their CDDP resistance by downregula-
`tion of glutathione S-transferase p expression (62). Furthermore,
`in a phase I study, the use of IL6 and GM-CSF in patients with renal
`cell carcinoma was associated with inverse clinical effects (63). We
`treated 18 MRCC patients with BE-8 mAb and demonstrated a
`clinical benefit, including abrogation of fever, hypercalcemia, and
`inflammatory syndrome, reduction of anemia and morphine
`intake, and weight increase, with objective responses observed
`(2/18 patients with partial responses, one with a minor response,
`and one with stable disease; ref. 12). In a phase I/II study,
`siltuximab was shown to stabilize the disease in more than
`50% of progressive MRCC patients, with one partial response
`and a favorable safety profile, a situation that could authorize
`further evaluation of dose-escalation strategies and/or combina-
`tion therapy (22). Nevertheless, to our knowledge, no further
`studies have been performed.
`
`Prostate cancer
`IL6 acts as a paracrine and autocrine growth factor for both
`benign and cancer prostate cells. The levels of IL6 and IL6R
`are increased during prostate carcinogenesis and tumor pro-
`gression (64). In addition, a correlation between increased
`serum IL6 and soluble IL6R levels with aggressiveness of the
`disease was reported (65). During the treatment of prostate
`cancer, castration resistance usually leads to the death of the
`patient. The signaling pathway mediated by IL6 represents an
`alternative pathway in the cancer-resistance phenotype acqui-
`sition and cancer progression (64, 65). During androgen dep-
`rivation therapy, a regulation loop may emerge between sex
`steroids and IL6, with a strong positive correlation with total-
`testosterone, androstenedione, and estradiol levels (66). Other
`gp130 cytokines have shown involvement in prostate tumor
`progression by promoting VEGF and urokinase plasminogen
`activator (67). A meta-analysis was recently performed to
`evaluate the association between the IL6 174GC genotype and
`susceptibility to prostate cancer with an increased risk observed
`in two cohort studies. Additional well-designed studies are
`needed to validate the role of IL6 genetic polymorphism in
`prostatic cancer risk (68).
`All the above-mentioned data show the importance of the
`IL6/IL6R pathway in the regulation of growth and drug resistance
`of prostate cancer cells. In a phase I study, 20 patients scheduled
`
`www.aacrjournals.org
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`Downloaded from
`
`clincancerres.aacrjournals.org
`
`Clin Cancer Res; 21(6) March 15, 2015
`
`on July 19, 2019. © 2015 American Association for Cancer Research.
`
`1253
`
`Lassen - Exhibit 1024, p. 6
`
`

`

`Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Rossi et al.
`
`Abbreviations:EU,EuropeanUnion;J,Japan;MCCD,MulticentricCastlemandisease;MM,multiplemyeloma;NR,notreported;RA,rheumatoidarthritis;Paraneop.,paraneoplastic.
`
`Tocilizumab
`
`þ
`
`þ
`
`þ
`
`NR
`NR
`NR
`
`NR
`
`
`
`þ
`
`þ
`
`No
`NR
`
`NR
`
`þ
`
`NR
`NR
`NR
`
`NR
`
`
`
`þ
`
`þ
`
`5.8(1.7–8.1)
`
`1.55(0.8–130)
`
`Crohndisease
`
`Prostatecancer
`
`No
`Paraneop.syndrome
`
`No
`Paraneop.syndromeeffectHCa
`
`No
`-effectHCa
`
`Tocilizumab(J;situximabEUandU.S.)
`þþ
`
`Tocilizumab
`þþ
`
`Drugregistration
`Diseaseimprovement
`
`þ
`
`þ
`
`NR
`NR
`
`þ
`
`þ
`
`þ
`
`þ
`
`withHIVinfection
`associated
`lymphoma
`
`Effecton
`
`29.2(0–129)
`
`Lymphoma
`
`þ
`
`þ
`
`Nomajorchange
`
`
`
`þ
`
`þ
`
`þ
`
`þ
`
`þ
`
`þ
`
`þ
`
`NR
`
`NR
`
`þ
`
`
`
`þ
`
`þ
`
`4.9(0.3–183)
`
`3.75(0–121)
`
`MRCC
`
`MM
`
`þ
`
`þ
`
`NR
`
`þ
`
`þ
`
`þ
`
`þ
`
`þ
`
`þ
`
`þ
`
`NR
`þþ
`NR
`
`þ
`
`þ
`
`þ
`
`þþ
`
`þþ
`
`17.6(0.1–181)
`
`5.6(2–19.4)
`
`MCCD
`
`RA
`
`1–8weeks
`(1.1–1.5g/dL)within
`Hemoglobinincrease
`Plateletdecrease(20%–30%decrease)
`Lymphocytecounts(peripheralblood)
`Analgesia
`Fever(Bsymptom)
`after3–4weeks)
`(20%–30%increase
`
`Albuminincrease
`
`2–4weeks)
`40%–70%reductionin
`20%decrease;other,
`proteins(ferritin
`Otheracute-phase
`within2–4days
`100%inhibition
`
`CRPinhibition90%–
`
`inhibitors
`
`EffectsofIL6
`
`(mg/L;range)
`
`MedianCRPlevels
`
`Table3.Clinicaleffectsofanti-IL6therapies(monoclonalantibodiestocilizumabandsiltuximab)
`
`1254
`
`Clin Cancer Res; 21(6) March 15, 2015
`
`Downloaded from
`
`clincancerres.aacrjournals.org
`
`on July 19, 2019. © 2015 American Association for Cancer Research.
`
`Clinical Cancer Research
`
`Lassen - Exhibit 1024, p. 7
`
`

`

`Published OnlineFirst January 14, 2015; DOI: 10.1158/1078-0432.CCR-14-2291
`
`Anti-IL6 Therapy
`
`for radical prostatectomy received either no drug or siltuximab
`(6 mg/kg, 5 patients/group with administration once, twice, and
`three times, prior to surgery). A decrease in phosphorylation of
`Stat3 transcription factor and p44/p42 MAPKs, with a down-
`regulation of genes immediately downstream of the IL6 signaling
`pathway and key enzymes of the androgen signaling pathway, was
`observed (69). A phase II study designed by SWOG was com-
`pleted in chemotherapy-pretreated patients with castration-resis-
`tant prostate cancer, showing stable disease in 7 patients (23%).
`An increase of IL6 was observed after treatment, likely due to the
`accumulation of stable IL6/anti-IL6 mAb complexes, as we dem-
`onstrated in other trials (16). This may explain the lack of clinical
`benefit (70). The combination of mitoxantrone/prednisone and
`siltuximab was not associated with a clinical improvement in a
`randomized phase II study, as compared with chemotherapy
`alone (23). Other molecules have been shown efficacious on
`tumorigenesis in prostate cancer,
`including polyphenols, by
`mediating a decrease in IL6 signaling (71). Because of a rise in
`IL6 both in vitro and in vivo in the presence of tyrosine kinase–
`resistant cells, IL6 may represent a biomarker for tyrosine kinase
`resistance (72).
`
`Other Potential Indications
`
`IL6 inhibition may have therapeutic indications in other can-
`cers, including ovarian cancer. Recently, a correlation between
`thrombocytosis,