660
`
`Ann Rheum Dis 2001;60:660–669
`
`EXTENDED REPORTS
`
`EVects of treatment with a fully human
`anti-tumour necrosis factor ♡ monoclonal
`antibody on the local and systemic homeostasis of
`interleukin 1 and TNF♡ in patients with
`rheumatoid arthritis
`
`P Barrera, L A B Joosten, A A den Broeder, L B A van de Putte, P L C M van Riel,
`W B van den Berg
`
`Abstract
`Objectives—To study the short term ef-
`fects of a single dose of D2E7, a fully
`human
`anti-tumour
`necrosis
`factor
`(TNF♡) monoclonal antibody (mAb), on
`the local and systemic homeostasis of
`interleukin 1♢ (IL1♢) and TNF♡ in patients
`with rheumatoid arthritis (RA).
`Methods—All patients with RA enrolled in
`a phase I, single dose, placebo controlled
`study with D2E7 in our centre were
`studied. Systemic cytokine levels, acute
`phase reactants, and leucocyte counts
`were studied at days 0, 1, and 14 after the
`first administration of anti-TNF mAb
`(n=39) or placebo (n=11). The cellularity
`and the expression of IL1 and TNF♡ in
`synovial tissue were studied in knee biopsy
`specimens obtained at baseline and at day
`14 in 25 consenting patients.
`Results—A single dose of anti-TNF mAb
`induced a rapid clinical improvement, a
`decrease in acute phase reaction, and
`increased lymphocyte counts in patients
`with active RA. The protein levels of IL1♢
`in the circulation were low and remained
`unchanged, but the systemic levels of IL1♢
`mRNA (p=0.002) and the concentrations
`of IL1 receptor antagonist (IL1ra) and IL6
`(p=0.0001) had already dropped within 24
`hours and this persisted up to day 14. Sys-
`temic levels of TNF♡ mRNA were low and
`remained unchanged, though total TNF♡
`(free and bound) in the circulation in-
`creased after D2E7, probably reflecting
`the presence of TNF-antiTNF mAb com-
`plexes (p<0.005, at days 1 and 14). Both
`TNF receptors dropped below baseline
`levels at day 14 (p<0.005). Despite clinical
`improvement of arthritis, no consistent
`immunohistological changes were seen
`two weeks after anti-TNF administration.
`Endothelial staining for IL1♢ tended to
`decrease in treated patients (p=0.06) but
`not in responders. The staining for IL1♢
`and TNF♡ in sublining layers and vessels
`
`www.annrheumdis.com
`
`was mutually correlated (rs=0.47 and 0.58
`respectively, p<0.0005) and the micro-
`scopic scores for inflammation correlated
`with sublining TNF♡ and IL1♢ scores
`(rs=0.65 and 0.54 respectively, p<0.0001),
`though none of these showed significant
`changes during the study.
`Conclusions—Blocking TNF♡ in RA re-
`sults in down regulation of IL1♢ mRNA at
`the systemic level and in reduction of the
`endogenous antagonists for IL1 and TNF
`and of other cytokines related to the acute
`phase response, such as IL6, within days.
`At the synovial level, anti-TNF treatment
`does not modulate IL1♢ and TNF♡ in the
`short term. The synovial expression of
`these cytokines does not reflect clinical
`response to TNF neutralisation.
`(Ann Rheum Dis 2001;60:660–669)
`
`In recent years, several approaches aimed at
`specific neutralisation of proinflammatory cyto-
`kines, such as interleukin 1 (IL1) and tumour
`necrosis factor (TNF), have proved successful
`in chronic inflammatory diseases, such as
`(RA)1–4 and Crohn’s
`rheumatoid arthritis
`disease.5 6
`In patients with RA, TNF has been targeted
`using monoclonal antibodies (mAb), either
`chimeric2 3 or humanised,4 and TNF receptor-
`fusion proteins.7 8 Some of these TNF antago-
`nists have shown their eYcacy in multicentre,
`placebo controlled trials and have been re-
`cently approved by the FDA. A potential draw-
`back of some TNF blocking agents is the
`development of human antichimeric antibod-
`ies, which may hamper or shorten the long
`term therapeutic eVects. This complication
`could be overcome using mAb devoid of
`murine regions.
`D2E7 (Knoll-BASF, Germany) is a fully
`human IgG1 anti-TNF♡ mAb with high
`specificity for recombinant and natural TNF♡,
`and it is generated with phage display tech-
`niques.9 Studies in more than 150 patients
`
`Department of
`Rheumatology,
`University Hospital,
`Nijmegen, The
`Netherlands
`P Barrera
`L A B Joosten
`A A den Broeder
`L B A van de Putte
`P L C M van Riel
`W B van den Berg
`
`Correspondence to:
`Dr P Barrera, Department of
`Rheumatology, University
`Hospital Nijmegen, PO Box
`9101, 6500 HB Nijmegen,
`The Netherlands
`p.barrera@reuma.azn.nl
`
`Accepted 25 October 2000
`
`Ex. 1004 - Page 1 of 15
`
`AMGEN INC.
`Exhibit 1004
`
`

`

`660
`
`Ann Rheum Dis 2001;60:660–669
`
`EXTENDED REPORTS
`
`EVects of treatment with a fully human
`anti-tumour necrosis factor ♡ monoclonal
`antibody on the local and systemic homeostasis of
`interleukin 1 and TNF♡ in patients with
`rheumatoid arthritis
`
`P Barrera, L A B Joosten, A A den Broeder, L B A van de Putte, P L C M van Riel,
`W B van den Berg
`
`Abstract
`Objectives—To study the short term ef-
`fects of a single dose of D2E7, a fully
`human
`anti-tumour
`necrosis
`factor
`(TNF♡) monoclonal antibody (mAb), on
`the local and systemic homeostasis of
`interleukin 1♢ (IL1♢) and TNF♡ in patients
`with rheumatoid arthritis (RA).
`Methods—All patients with RA enrolled in
`a phase I, single dose, placebo controlled
`study with D2E7 in our centre were
`studied. Systemic cytokine levels, acute
`phase reactants, and leucocyte counts
`were studied at days 0, 1, and 14 after the
`first administration of anti-TNF mAb
`(n=39) or placebo (n=11). The cellularity
`and the expression of IL1 and TNF♡ in
`synovial tissue were studied in knee biopsy
`specimens obtained at baseline and at day
`14 in 25 consenting patients.
`Results—A single dose of anti-TNF mAb
`induced a rapid clinical improvement, a
`decrease in acute phase reaction, and
`increased lymphocyte counts in patients
`with active RA. The protein levels of IL1♢
`in the circulation were low and remained
`unchanged, but the systemic levels of IL1♢
`mRNA (p=0.002) and the concentrations
`of IL1 receptor antagonist (IL1ra) and IL6
`(p=0.0001) had already dropped within 24
`hours and this persisted up to day 14. Sys-
`temic levels of TNF♡ mRNA were low and
`remained unchanged, though total TNF♡
`(free and bound) in the circulation in-
`creased after D2E7, probably reflecting
`the presence of TNF-antiTNF mAb com-
`plexes (p<0.005, at days 1 and 14). Both
`TNF receptors dropped below baseline
`levels at day 14 (p<0.005). Despite clinical
`improvement of arthritis, no consistent
`immunohistological changes were seen
`two weeks after anti-TNF administration.
`Endothelial staining for IL1♢ tended to
`decrease in treated patients (p=0.06) but
`not in responders. The staining for IL1♢
`and TNF♡ in sublining layers and vessels
`
`www.annrheumdis.com
`
`was mutually correlated (rs=0.47 and 0.58
`respectively, p<0.0005) and the micro-
`scopic scores for inflammation correlated
`with sublining TNF♡ and IL1♢ scores
`(rs=0.65 and 0.54 respectively, p<0.0001),
`though none of these showed significant
`changes during the study.
`Conclusions—Blocking TNF♡ in RA re-
`sults in down regulation of IL1♢ mRNA at
`the systemic level and in reduction of the
`endogenous antagonists for IL1 and TNF
`and of other cytokines related to the acute
`phase response, such as IL6, within days.
`At the synovial level, anti-TNF treatment
`does not modulate IL1♢ and TNF♡ in the
`short term. The synovial expression of
`these cytokines does not reflect clinical
`response to TNF neutralisation.
`(Ann Rheum Dis 2001;60:660–669)
`
`In recent years, several approaches aimed at
`specific neutralisation of proinflammatory cyto-
`kines, such as interleukin 1 (IL1) and tumour
`necrosis factor (TNF), have proved successful
`in chronic inflammatory diseases, such as
`(RA)1–4 and Crohn’s
`rheumatoid arthritis
`disease.5 6
`In patients with RA, TNF has been targeted
`using monoclonal antibodies (mAb), either
`chimeric2 3 or humanised,4 and TNF receptor-
`fusion proteins.7 8 Some of these TNF antago-
`nists have shown their eYcacy in multicentre,
`placebo controlled trials and have been re-
`cently approved by the FDA. A potential draw-
`back of some TNF blocking agents is the
`development of human antichimeric antibod-
`ies, which may hamper or shorten the long
`term therapeutic eVects. This complication
`could be overcome using mAb devoid of
`murine regions.
`D2E7 (Knoll-BASF, Germany) is a fully
`human IgG1 anti-TNF♡ mAb with high
`specificity for recombinant and natural TNF♡,
`and it is generated with phage display tech-
`niques.9 Studies in more than 150 patients
`
`Department of
`Rheumatology,
`University Hospital,
`Nijmegen, The
`Netherlands
`P Barrera
`L A B Joosten
`A A den Broeder
`L B A van de Putte
`P L C M van Riel
`W B van den Berg
`
`Correspondence to:
`Dr P Barrera, Department of
`Rheumatology, University
`Hospital Nijmegen, PO Box
`9101, 6500 HB Nijmegen,
`The Netherlands
`p.barrera@reuma.azn.nl
`
`Accepted 25 October 2000
`
`Ex. 1004 - Page 2 of 15
`
`

`

`Anti-TNF mAb eVects on IL1♢ and TNF♡
`
`661
`
`show that repeated intravenous or subcutane-
`ous administration of D2E7 is safe and results
`in rapid clinical improvement in patients with
`active RA.10–13
`Several studies with another IgG1 anti-TNF
`mAb have clearly shown that blocking TNF
`reduces the acute phase reaction and decreases
`the local and systemic levels of adhesion
`molecule in patients with RA.14–17 In vitro stud-
`ies have also shown that neutralisation of TNF
`reduces the production of IL1 in synovial cul-
`tures.18 Whether such treatments also down
`regulate the synovial expression of IL1 and
`TNF in RA has not yet been fully elucidated.
`Observations in small numbers of patients sug-
`gest that this might be the case.14 19 20 In the
`present study we investigated the short term
`eVects of the first dose of D2E7 or placebo on
`the homeostasis of the two main proinflamma-
`tory cytokines IL1 and TNF at the systemic
`and the synovial level.
`
`Patients and methods
`PATIENTS
`Patients with RA according to the American
`College of Rheumatology criteria21 and with
`active disease, defined by a disease activity
`score (DAS)22 >3.2, who were enrolled in a
`double blind, multicentre clinical trial with
`anti-TNF♡ monoclonal antibody (D2E7) at
`our centre, were studied. Disease modifying
`antirheumatic drug treatment was withheld
`and non-steroidal anti-inflammatory drugs or
`low dose oral steroids (<10 mg/day), or both,
`were kept constant in a three week wash out
`period before and during the study. Patients
`were randomly assigned to receive 0.5, 1, 3, 5,
`or 10 mg/kg of D2E7 and each dose group
`included two patients treated with placebo,
`who received active drug at six weeks if they
`still fulfilled the entry criteria.11 13
`D2E7 or placebo was given as a slow
`intravenous infusion over three to five minutes.
`The preparation consists of a non-pyrogenic
`solution of 25 mg/ml D2E7 mAb in 1.2%
`
`mannitol, 0.12% citric acid, 0.02% sodium cit-
`rate. The placebo consists of the same ingredi-
`ents, except that D2E7 is excluded.
`Results of the multicentre study including
`120 patients in three centres show that clinical
`response is already apparent within 24 hours
`and maximal between one and two weeks after
`D2E7 administration.11 13 The clinical eVect is
`maximal at a dose of 1 mg/kg and shows a pla-
`teau in the dose-response curve thereafter.11 13
`Therefore,
`for the aims of
`this study, we
`focused on the short term eVects seen during
`the first two weeks after infusion of D2E7/
`placebo and subdivided the patients into three
`dose groups consisting of placebo (n=11), 0.5
`mg/kg (n=8), and 1–10 mg/kg D2E7 (n=31).
`
`CONCENTRATIONS AND GENE EXPRESSION OF
`CYTOKINES IN PERIPHERAL BLOOD
`Blood samples were drawn at 8–9 am on days
`0, 1, and 14 after infusion in endotoxin-free
`Vacutainer tubes with 15% EDTA-K3 (Becton
`and Dickinson, Rutherford, NJ). Samples were
`directly centrifuged (2250 g, 10 minutes and
`15 000 g, 5 minutes) to assess circulating
`cytokine
`concentrations
`in
`platelet-free
`plasma. Aliquots were stored at −20(cid:176) C until
`assay.
`IL1♢ was measured using a high sensitivity
`enzyme linked immunosorbent assay (ELISA;
`sensitivity 0.1 pg/ml; Quantikine HS, R&D,
`Minneapolis, USA) according to the manufac-
`turer’s instructions. Interleukin 1 receptor
`antagonist (IL1ra) was measured by radioim-
`munoassay (sensitivity 40 pg/ml; interassay and
`intra-assay variation <10%, respectively). IL6
`and both soluble TNF receptors (sTNFR)
`were measured by ELISA (sensitivity 8 pg/ml
`and 0.1 ng/ml
`respectively) as previously
`described.23 24 Total TNF♡ was measured by
`ELISA (sensitivity 10 pg/ml). The latter had
`been validated in spiking experiments showing
`adequate TNF♡ recovery and no hampering by
`therapeutic concentrations of D2E7 (range
`25–250 mg/l).11
`
`Table 1 Baseline characteristics and response percentages in this study according to the dose group
`
`Placebo (n=11)
`
`0.5 mg/kg (n=8)
`
`1–10 mg/kg (n=31)
`
`p Value
`
`Age, mean (SD) (years)
`Female (%)
`RF* positivity (%)
`Disease duration, median (p25–p75) (years)
`Previous DMARDs*, mean (SD) (n)
`Prednisone (%)
`DAS*, mean (SD)
`Ritchie articular index, mean (SD)
`Swollen joint count, mean (SD)
`ESR*, median (p25–p75) (mm/1st h)
`CRP*, median (p25–p75) (mg/l)
`Haemoglobin, mean (SD) (mmol/l)
`Leucocytes, median (p25–p75) (· 109/l)
`Polymorphonuclear cells
`Lymphocytes
`Monocytes
`Platelets, mean (SD) (· 109/l)
`Responders† at (%)
`2 Weeks
`12 Weeks
`24 Weeks
`
`60 (8)
`73
`100
`13 (4–23)
`4.5 (1.6)
`72
`4.8 (1.1)
`22 (13)
`18 (5)
`24 (7–39)
`23 (6–90)
`7.6 (0.9)
`8.1 (6.3–10.5)
`5.77 (4.76–8.12)
`1.16 (0.85–1.60)
`0.52 (0.40–0.58
`329 (48)
`
`53 (18)
`75
`100
`8 (4–15)
`4.5 (2.6)
`38
`5.2 (1.3)
`25 (11)
`18 (5)
`15 (9–38)
`22 (12–42)
`7.4 (1.2)
`7.5 (6.5–8.9)
`5.07 (4.55–6.51)
`1.37 (1.08–2.26)
`0.42 (0.38–0.49)
`306 (65)
`
`57 (14)
`55
`97
`10 (5–19)
`5 (1.7)
`52
`5.2 (1)
`26 (11)
`18 (6)
`36 (23–55)
`63 (33–115)
`7.2 (1.0)
`7.2 (5.9–10.4)
`4.78 (4.05–8.16)
`1.14 (0.85–1.31)
`0.46 (0.38–0.63)
`377 (130)
`
`9
`—
`—
`
`37.5
`50
`87.5
`
`65‡
`76.6
`70
`
`0.05
`0.05
`
`*RF = rheumatoid factor; DMARDs = disease modifying antirheumatic drugs; DAS = disease activity score; ESR = erythrocyte
`sedimentation rate; CRP = C reactive protein.
`†Response according to the EULAR criteria for moderate and good response.
`‡16% of the patients showed clinical response already at 24 hours after 1–10 mg/kg D2E7 administration.
`
`www.annrheumdis.com
`
`Ex. 1004 - Page 3 of 15
`
`

`

`Barrera, Joosten, den Broeder, et al
`
`at 42(cid:176) C, and 10 minutes at 95(cid:176) C in a Master-
`cycler 5330 (Eppendorf, Hamburg, Germany).
`PCR reaction mixtures consisted of 3 µl cDNA
`in 50 µl PCR buVer (20 mM Tris-HCl pH 8.4,
`50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin)
`containing 100 µM dNTPs, 1.25 U Taq
`polymerase, and 0.3 µM of each primer.25 A
`total of 29 PCR cycles (denaturation 30
`seconds at 92(cid:176) C, annealing 30 seconds at
`55(cid:176) C, and extension 90 seconds at 72(cid:176) C) were
`IL1♢ mRNA and TNF♡
`performed for
`mRNA, and 24 cycles were performed for ♢
`2
`microglobulin. PCR products underwent elec-
`trophoresis on 2% agarose gel and were stained
`with ethidium bromide. Gels were scanned on
`a densitometer and analysed using the Molecu-
`lar Analyst software. Results are expressed as a
`IL1♢ or TNF♡ mRNA to the
`ratio of
`housekeeping gene.
`
`SYNOVIAL BIOPSIES AND
`IMMUNOHISTOCHEMISTRY
`Percutaneous synovial biopsy specimens of
`the knee were obtained with a Parker Pearson
`needle at baseline and 14 days after the first
`dose of D2E7/placebo in all consenting
`patients. Biopsy was preceded by knee joint
`examination for tenderness (0=none, 1=re-
`sponse on questioning, 2=spontaneous re-
`sponse, 3=withdrawal) and swelling (0=none,
`1=thickening without loss of bony contours;
`bulge sign, 2=loss of bony contours; palpable
`but not tightly distending eVusions, 3=bulging
`synovial proliferation; tightly distending eVu-
`sions). Macroscopic knee joint scores were cal-
`culated by adding up the tenderness and swell-
`ing scores. An average of 30 biopsy specimens
`was obtained at each occasion and immediately
`fixed in 10% formalin and embedded in paraf-
`fin. Serial 7 µM microtome sections were
`mounted on superfrost slides and either stained
`with haematoxylin and eosin (H&E) or used
`for histochemical staining as previously de-
`scribed.26 27 For each marker, all sections were
`stained in the same run to minimise interassay
`variations. Slides were incubated with anti-
`TNF♡ (IgG1, Monosan, Uden, The Nether-
`lands) or anti-IL1♢ mAb (IgM, 12E9, Onco-
`gene Science, Manhasset, NY, USA). This
`primary step was followed by incubation with
`normal horse serum and with biotinylated
`horse antimurine IgG. Slices were stained with
`avidin peroxidase (Elite kit, Vector, Burlin-
`game, CA), developed with diaminobenzidine
`and counterstained with haematoxylin for
`three minutes. Controls consisted of
`(a)
`irrelevant primary isotype-specific IgG1 and
`IgM antibodies obtained from normal horse
`serum, and (b) omission of the secondary anti-
`bodies.
`All areas of each section were randomly ana-
`lysed by two “blinded” observers using semi-
`quantitative five point scales. H&E sections
`were scored for the presence of lymphocytes,
`plasma cells, and polymorphonuclear cells with
`a scale ranging from 0 (no or minimal infiltra-
`tion) to 4 (abundant inflammatory infiltrate),
`the lining hyperplasia was also scored (0=1–2
`layers, 1=3–4 layers, 2=5–6 layers, and 3=more
`than 6 layers). An “inflammation score” was
`
`Aliquots of 500 µl blood for RNA isolation
`were mixed with guanidinium isothiocyanate at
`a ratio of 1:1 and stored at −70(cid:176) C. Isolation of
`whole blood mRNA and reverse transcriptase
`polymerase chain reaction (RT-PCR) analysis
`were performed as previously described by
`Netea et al.25 Briefly, aliquots of 0.5 µg total
`RNA were diluted in 20 µl RT buVer (50 mM
`Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2)
`containing 10 mM dithiothreitol, 5 µM ran-
`dom hexamers, 250 µM dNTPs, 20 U RNAsin,
`and 200 M MLV RT. RT reaction was
`performed for 10 minutes at 20(cid:176) C, 45 minutes
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`662
`
`A
`
`8 7 6 5
`
`34
`
`2 1
`
`80
`
`60
`
`40
`
`20
`
`0
`
`B
`
`C
`
`160
`
`120
`
`80
`
`40
`
`DAS
`
`ESR (mm/1st h)
`
`CRP (mg/l)
`
`20
`
`0
`
`14
`
`0
`
`1
`1
`14
`1
`Placebo
`0.5 mg/kg
`1–10 mg/kg
`Figure 1 (A) Disease activity score (DAS), (B) erythrocyte sedimentation rate (ESR),
`and (C) C reactive protein (CRP) in patients receiving a single dose of placebo or 0.5
`mg/kg or 1–10 mg/kg anti-tumour necrosis factor monoclonal antibody. Boxes represent the
`25th, 50th, and 75th centiles; vertical lines indicate the 5th and 95th centiles.
`Measurements on days 0, 1, and 14 after infusion (x axis). Comparisons versus baseline
`shown as *p<0.05, **p<0.005, and ***p<0.0005.
`
`14
`
`0
`
`www.annrheumdis.com
`
`Ex. 1004 - Page 4 of 15
`
`

`

`Anti-TNF mAb eVects on IL1♢ and TNF♡
`
`663
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`14
`1
`Placebo
`
`0
`14
`1
`0.5 mg/kg
`
`0
`14
`1
`1–10 mg/kg
`
`0
`
`1
`Placebo
`
`14
`
`0
`14
`1
`1–10 mg/kg
`
`B
`
`D
`
`300
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`9 8 7 6 5 4 3 2
`
`F
`
`1
`0.9
`0.8
`0.7
`0.6
`0.5
`0.4
`0.3
`0.2
`0.1
`0
`
`IL6 (pg/ml)
`
`p75 sTNFR (ng/ml)
`
`TNF/b2M ratio
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`
`1
`
`14
`
`0
`14
`1
`Placebo
`
`0
`14
`1
`0.5 mg/kg
`
`0
`14
`1
`1–10 mg/kg
`
`0
`
`1
`Placebo
`
`14
`
`0
`14
`1
`1–10 mg/kg
`
`A
`
`1100
`
`900
`
`700
`
`500
`
`300
`
`100
`
`IL1ra (pg/ml)
`
`C
`
`5 4 3 2 1 0
`
`E
`
`2.5
`2.3
`2.1
`1.9
`1.7
`1.5
`1.3
`1.1
`0.9
`0.7
`0.5
`
`p55 sTNFR (ng/ml)
`
`IL1b/b2M ratio
`
`Figure 2 Circulating concentrations of (A) interleukin 1 receptor antagonist (IL1ra), (B) IL6, (C) 55th centile (p55),
`and (D) 75th centile (p75) soluble tumour necrosis factor receptors (sTNFR). (E) IL1♢ and (F) TNF♡ mRNA in whole
`blood normalised for the presence of ♢
`
`2 microglobulin (♢2M). Boxes represent 25th, 50th, and 75th centiles; vertical lines
`indicate the 5th and 95th centiles. Measurements on days 0, 1, and 14 after infusion (x axis). Comparisons versus baseline
`shown as *p<0.5, **p<0.005, and ***p<0.0005.
`calculated by adding these four components
`(range 0–15), as previously described by Tak et
`al.14 The staining for IL1 and TNF in lining,
`sublining, and vessels was also scored semi-
`quantitatively on a five point scale (0 to 4). Dif-
`ferences in readings of one point were taken as
`the average, diVerences exceeding one point
`were resolved by mutual agreement.
`
`Results
`CLINICAL RESULTS, ACUTE PHASE REACTION, AND
`LEUCOCYTE SUBSETS
`Table 1 summarises the baseline characteristics
`of the patients included in the present study.
`Patients were subdivided into groups receiving
`placebo, 0.5 mg/kg D2E7, and 1–10 mg/kg
`D2E7 because, as previously mentioned, the
`dose-response curve in the multicentre study
`(n=120) showed a plateau at doses of 1 mg/kg
`D2E7.11 13
`Administration of D2E7, but not placebo,
`rapidly reduced disease activity as measured by
`the DAS, a composite disease activity score22 (fig
`1A), and its individual components, including
`swollen and tender joint counts, patient well-
`being (data not shown), and erythrocyte sedi-
`mentation rate (ESR; fig 1B). In the group
`treated with 1–10 mg/kg D2E7, the decrease in
`
`STATISTICAL ANALYSIS
`Analysis was performed using the SAS statistical
`package (SAS 6.04 PC version). Data are
`expressed as means (SD) or as median (25th
`(p25)–75th (p75)
`centile)
`if
`appropriate.
`Within-group comparisons were analysed by
`paired Student’s t or Mann Whitney rank sum
`test. Baseline comparisons between groups were
`performed using one way analysis of variance or
`Kruskal-Wallis tests. Correlations are expressed
`using Spearman’s rank correlation coeYcient.
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`Barrera, Joosten, den Broeder, et al
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`increased in the placebo group (figs 1B and C).
`The 0.5 mg/kg group, which included fewer
`patients (n=8), also showed a clear decrease in
`ESR and CRP after two weeks.
`Baseline white blood cell and leucocyte
`subset counts were similar in all groups (table
`1). Total white blood cell counts did not
`change during the study (data not shown), but
`a rise in day 1 lymphocyte counts was seen in
`all groups, being more marked in patients
`receiving 1–10 mg/kg D2E7 (median 14.5%
`before infusion v 22% at day 1; p<0.01). In this
`group only, the lymphocyte counts were still
`higher than baseline at day 14. Conversely,
`there was a drop in day 1 polymorphonuclear
`cell counts in all groups, which only reached
`significance in the 1–10 mg/kg group at day 1
`(median 77% at baseline v 66.5% at day 1;
`p=0.02).
`
`SYSTEMIC CYTOKINE MEASUREMENTS
`Baseline IL1♢ and TNF♡ levels were below or
`around the detection limit of the radioimmuno-
`assays currently used in our laboratory and
`similar to those found in healthy subjects (data
`not shown). A high sensitivity ELISA (Quan-
`tikine HS, R&D, Minneapolis, USA) showed
`that IL1♢ protein levels were low and did not
`change significantly after the first dose of D2E7
`or placebo (median levels at baseline, day 1,
`and day 14 were 0.55, 0.4, and 0.5 pg/ml after
`1–10 mg/kg D2E7 and remained at 0.6 pg/ml
`before and after placebo administration).
`Mean baseline TNF♡ levels
`(free and
`bound) measured by ELISA did not change
`after infusion of placebo but increased after
`D2E7 administration (41 pg/ml at baseline v
`76 and 63 pg/ml at days 1 (p<0.0001) and 14
`(p<0.01), respectively), which may reflect the
`formation of circulating TNF-anti-TNF com-
`plexes.
`To complement the data at the protein level,
`systemic IL1♢ and TNF♡ mRNA levels in
`whole blood were measured in the placebo and
`1–10 mg/kg D2E7 group by RT-PCR. Baseline
`IL1♢/♢
`2 microglobulin ratios in patients with
`RA were raised compared with those in healthy
`controls (median (range) 1.45 (0.57–2.95) v
`0.35 (<0.10–0.69)). As shown in fig 2, IL1♢
`mRNA expression decreased within 24 hours
`after D2E7 administration (p=0.002) and
`remained lower
`than baseline at day 14
`(p=0.007), whereas no significant changes
`occurred after placebo. Systemic TNF♡
`mRNA levels were lower than those of IL1♢,
`overlapped largely with those found in healthy
`controls (median (range) 0.13 (<0.10–0.85) in
`RA v <0.10 (<0.10–0.30) in controls), and
`remained unchanged during the study. Sys-
`temic levels of IL1♢ and TNF♡ mRNA were
`not correlated (rs (95%CI)=0.13 (−0.18 to
`0.43)).
`IL1ra and IL6
`The concentrations of
`decreased sharply after D2E7 administration
`(p<0.0001 and <0.005 at days 1 and 14,
`respectively, in the 1–10 mg/kg group). Both
`sTNFR types decreased also after treatment. In
`patients receiving 1–10 mg/kg D2E7, the p75
`levels dropped from median baseline values
`of 5 ng/ml to 4.3 (p<0.005) and 3.7 ng/ml
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`DAS
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`Macroscopic knee score
`
`Histological score for inflammation
`
`Figure 3 (A) Disease activity score (DAS), (B) pain and swelling scores, and (C)
`histological scores for inflammation in 25 patients undergoing serial biopsies. Time in weeks
`and placebo (n=8) or anti-tumour necrosis factor (anti-TNF) treatment (n=17) shown in
`the x axis. Scores for more than one patient (·
`exact number) shown as bold lines.
`
`DAS was already significant within 24 hours
`after the infusion (mean (SD) 4.87 (0.79) v 5.25
`(0.95); p<0.002) and reached a nadir at week 2
`(4.06 (0.85), p<0.0001). The 0.5 mg/kg group
`showed a drop in DAS values significant at week
`2 (p<0.01). At this time point, 20/31 (65%) and
`3/8 (38%) of the patients treated with 1–10 and
`0.5 mg/kg D2E7, respectively,
`fulfilled the
`EULAR criteria for clinical response28 in con-
`trast with only 1/11 (9%) patients receiving pla-
`cebo. Five (16%) patients in the 1–10 mg/kg
`group showed clinical response already 24 hours
`after infusion.
`Baseline acute phase reaction parameters,
`such as the ESR, C reactive protein (CRP), and
`platelet counts tended to be higher in the 1–10
`mg/kg group (table 1) and were also signifi-
`cantly decreased two weeks after administra-
`tion of anti-TNF mAb but unchanged or
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`Anti-TNF mAb eVects on IL1♢ and TNF♡
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`665
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`Figure 4 Immunohistochemical staining in the same patient at baseline ((A) tumour necrosis factor ♡ (TNF♡), (B)
`interleukin 1♢ (IL1♢)) and 14 days after ((C) TNF♡, (D) IL1♢) the first dose of anti-TNF♡. Note the unchanged
`expression of cytokines in the synovial lining (s, arrows), sublining, and blood vessels (bv). No IL1♢ or TNF♡ staining was
`seen in controls with (E) an irrelevant primary antibody and (F) when the secondary antibody was omitted. Original
`magnification · 200.
`(p=0.0001) after 1 and 14 days respectively
`and the p55 sTNFR levels fell from median
`baseline values of 3.1 ng/ml to 2.5 ng/ml at day
`14 (p<0.002) (fig 2).
`Positive correlations between acute phase
`reactants and the endogenous antagonists of
`IL1 and TNF were found: CRP measurements
`
`correlated with the levels of IL1ra (rs=0.47;
`p<0.001), p55 sTNFR (rs=0.66; p<0.0001)
`and IL6 (rs=0.65; p<0.0001), whereas ESR
`levels correlated with p75 sTNFR (rs=0.51;
`p<0.002). In contrast, neither TNF♡ nor IL1♢,
`at protein or mRNA level, were related to acute
`phase parameters.
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`IL1 and TNF lining
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`IL1 and TNF sublining
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`IL1 and TNF vessels
`
`Anti-TNF
`Placebo
`Placebo
`Anti-TNF
`Figure 5 Semiquantitative scores for interleukin 1♢ (IL1♢; left columns) and tumour necrosis factor ♡ (TNF♡; right
`columns). Staining in (A) lining, (B) sublining, and (C) vessels in biopsy specimens taken at baseline and at day 14 after
`administration of placebo (n=8) or anti-TNF treatment (n=17). Scores for more than one patient (·
`exact number) shown
`as bold lines.
`
`IMMUNOHISTOLOGICAL FINDINGS
`A total of 25 patients underwent synovial biop-
`sies at baseline and 14 days after infusion of
`placebo (n=8) or 1–10 mg/kg D2E7 (n=17). At
`this time in the treated group, the DAS had
`dropped from 5.0 (1.0)
`to 4.0 (1.1)
`(p<0.0005), and 11 patients
`fulfilled the
`EULAR criteria for clinical response.28 Con-
`versely, DAS scores remained unchanged or
`increased (4.8 (1.3) at baseline v 5.1 (1.6) after
`14 days) in the placebo group, where only one
`patient fulfilled the EULAR response criteria28
`(fig 3A). The macroscopic joint scores for
`swelling and pain at the biopsied joint were
`unchanged two weeks after placebo but de-
`creased in treated patients (p=0.02; fig 3B).
`The microscopic scores for inflammation14
`showed a more scattered pattern and no
`
`significant changes in either group (fig 3C).
`Baseline microscopic inflammation scores were
`weakly correlated with the macroscopic scores
`for pain and swelling (rs=0.3; p<0.05).
`TNF♡ and IL1♢ staining were seen in all
`layers of most biopsy specimens. The scores
`for both cytokines in the lining, sublining, and
`especially in vessels were mutually correlated
`(rs=0.43; p=0.002 for the lining and rs=0.47
`and 0.58; p<0.0005 for the sublining and ves-
`sels, respectively). Nevertheless, some diVer-
`ences were found in the staining patterns of
`both cytokines: TNF showed a more dense
`and intense staining in sublining layers,
`lymphocyte aggregates, and vessels than IL1,
`which tended to be more diVuse (fig 4). The
`microscopic scores for
`inflammation were
`positively correlated with the scores for TNF
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`Anti-TNF mAb eVects on IL1♢ and TNF♡
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`667
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`and IL1 in the sublining (rs=0.65 and 0.54
`respectively; p<0.0001) and to a lesser extent
`with the TNF staining in the lining (rs=0.38;
`p=0.005).
`Figure 5 shows the individual synovial scores
`for IL1 and TNF after administration of
`anti-TNF mAb or placebo. As shown, there
`was a marked interindividual and intra-
`individual variation in the synovial staining for
`these cytokines. Decreases greater than one
`point in the scores for IL1♢ or TNF♡ were rare
`among placebo treated patients. On the other
`hand, both increased and decreased scores for
`IL1♢ and TNF♡ were detected in the lining
`and sublining of patients treated with anti-
`TNF. With the exception of a trend towards a
`decrease in the IL1♢ staining in vessels among
`the treated patients (p<0.06), no significant
`changes were seen.
`Additionally, analysis was performed accord-
`ing to the clinical response achieved at day 14
`after infusion. At this time point, a total of 12
`patients (all but one in the group treated with
`anti-TNF) were responders according to the
`EULAR criteria28 (DAS decreased from 4.9
`(1.1) to 3.4 (0.6); p<0.0001), whereas 13
`patients, including seven who had received pla-
`cebo, were non-responders (DAS 5.1 (1.2) v
`5.2 (1.2)). Macroscopic knee joint score for
`swelling and pain dropped from 2 (2.3) to 0.8
`(1.1) (p<0.05) among responders but re-
`mained unchanged (2.2 (2.0) v 2.2 (2.0)) in
`the non-responders.
`In contrast with the clinical improvement
`observed in responders, the microscopic in-
`flammation scores (H&E) and the staining for
`IL1♢ or TNF♡ in lining, sublining, and vessels
`were not significantly changed from baseline
`compared with day 14 (data not shown). Sub-
`group analysis taking into account the indi-
`vidual dose of D2E7 given gave the same
`results.
`
`Discussion
`Our study shows that in patients with active
`RA, a single dose of human anti-TNF antibody
`is clinically eVective and results in rapid down
`regulation of IL1♢ mRNA at the systemic level.
`Such eVect was seen in unstimulated condi-
`tions, using a sensitive PCR, normalised for the
`presence of ♢
`2 microglobulin, which corrects
`for fluctuations in peripheral blood mononu-
`clear cell (PBMC) counts. The inhibition of
`IL1 by anti-TNF mAb treatment occurred at
`the transcriptional
`level and seemed rather
`specific. TNF neutralisation did not alter the
`levels of TNF♡ message in the circulation, and
`no change in IL1♢ or TNF♡ mRNA expression
`was seen in the placebo group.
`The production of both IL1 and TNF is
`transcriptionally regulated29 30 and both cyto-
`kines share many proinflammatory eVects.
`Therefore, a decrease in IL1 message might
`play a part in at least some of the downstream
`changes seen after treatment with this and
`other anti-TNF mAb. These include inhibition
`of IL6 and acute phase reaction, chemokines
`and nitric oxide production, and a decrease in
`angiogenesis, endothelial activation, and carti-
`lage breakdown markers16 17 31
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`In our study, baseline mRNA levels of IL1♢
`in whole blood were raised in patients with RA
`compared with normal controls. Raised levels
`of mRNA for IL1 and other proinflammatory
`cytokines such as IL8 have also been measured
`in isolated PBMC of patients with RA,32 33 and
`these may reflect an increased activation status
`of PBMC in the circulation. Systemic TNF
`mRNA levels were lower, and did not correlate
`with those of IL1 mRNA, which is in line with
`the earlier reported diVerential regulation of
`IL1 and TNF in PBMC30 32 and in whole
`blood.29
`Inhibition of the synthesis of IL1♢ after
`blocking TNF has been shown to occur in
`rheumatoid synovial cultures.18 34 Direct evi-
`dence for such an eVect in patients with RA has
`been hard to find because, as we and others
`have shown,17 protein levels of IL1♢ in the cir-
`culation are mostly low or undetectable.
`I