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`Clin Exp Immunol 2002; 130:75–84
`
`
`
`Blackwell Science, Ltd
`CEIClinical and Experimental Immunology
`Oxford, UK
`0009-9104Blackwell Publishing Ltd, 2002
`130
`
`Original Article
`
`J. B. Marriott
`
`et al.
`
`Diverse effects of Thd and its analogues on TNF-
`α
`
`/TNFR2 during T cell co-stimulation
`
`αααα
`Thalidomide and its analogues have distinct and opposing effects on TNF-
` and
`TNFR2 during co-stimulation of both CD4
` and CD8
` T cells
`+
`+
`
`J. B. MARRIOTT, I. A. CLARKE, K. DREDGE, G. MULLER*, D. STIRLING* & A. G. DALGLEISH
`*
`Division of Oncology, Department of OGEM, St George’s Hospital Medical School, London, UK, and
`Celgene Corporation,
`Warren, New Jersey, USA
`
`(Accepted for publication 28 June 2002)
`
`SUMMARY
`
`Thalidomide (Thd) is clinically useful in a number of conditions where its efficacy is probably related to
`α
` activity. More recently, Thd has also been shown to co-stimulate T cells and second gen-
`its anti-TNF
`-
`eration co-stimulatory (IMiD™) analogues are currently being assessed in the treatment of cancer
`patients. However, in contrast to their known suppressive effects during inflammatory stimuli, the
`α
` and TNF receptors (TNFRs) during T cell co-stimulation are not
`effects of Thd/IMiDs on TNF-
`known. We sought to determine the effect of Thd, two clinically relevant IMiDs (CC-4047, ACTIMID™
`α
` production
`and CC-5013, REVIMID™) and a non-stimulatory SelCID analogue (CC-3052) on TNF-
`and on the expression and shedding of TNFRs during co-stimulation. We found that co-stimulation of
`α
`CD3-induced T cell surface expression of TNFR2
`PBMC with Thd/IMiDs, but not CC-3052, prevented
`and thereby reduced soluble TNFR2 (sTNFR2) levels. However, there was no effect on total (surface/
`intracellular) TNFR2 protein expression, suggesting inhibition of trafficking to the cell membrane. The
`α
` produc-
`extent of co-stimulation by Thd/IMiDs (assessed by CD69/CD25 expression and IL-2/sIL-2R
`+
`+
` and CD8
` T lymphocytes and correlated with TNFR2 inhibition. Co-
`tion) was similar for CD4
`stimulation, but not the early inhibitory effect on TNFR2, was IL-2-dependent and led to increased
`α
`+
`+
` production by both CD4
` and CD8
` T lymphocytes. The clinical relevance of this observation
`TNF-
`α
` during REVIMID™ treatment of patients with
`was confirmed by the elevation of serum TNF-
`advanced cancer. Together, these results suggest a possible role for TNF-mediated events during co-
`stimulation and contrast with the TNF inhibitory effects of Thd and its analogues during inflammatory
`stimuli.
`
`Keywords
`
`co-stimulation immunomodulation thalidomide T lymphocyte TNF
`
`INTRODUCTION
`
`Thalidomide (Thd) is emerging as an important immunothera-
`peutic drug in a number of clinical situations [1–4]. It is well estab-
`α
` agent in the context of monocyte/
`lished as an anti-TNF-
`α
` [5,6] and clinically during TNF-
`-
`macrophage activation
`in vitro
`mediated inflammatory disease [6]. However, other immunomod-
`ulatory properties may explain the extensive range of its clinical
`activity. More recently, it has been shown to inhibit monocyte IL-
`12 production [7] and also to be able to induce the activation and
`+
` T cells stimulated via the T cell receptor in
`proliferation of CD8
`the absence of co-stimulation [8]. Thus, in certain clinical settings,
`such as in the treatment of cancer patients, Thd may act as an
`
`Correspondence: Dr J. B. Marriott, Division of Oncology, St George’s
`Hospital Medical School, Cranmer Terrace, Tooting, London, SW17 ORE,
`UK.
`E-mail: jmarriot@sghms.ac.uk
`
`© 2002 Blackwell Publishing Ltd
`
`adjuvant to promote T-cell responses. However, Thd treatment
`α
` [9–
`has also been known to increase circulating levels of TNF-
`12] and IL-12 [13] and it appears likely that the effects of Thd vary,
`depending on the relative predominance of monocyte/macroph-
`age and T cell activation. Furthermore, the effects of Thd may also
`vary depending on the activation of particular signalling pathways
`[14].
`Significant side effects such as somnolence and peripheral
`neuropathy are associated with clinically effective Thd treatment.
`However, the design and synthesis of Thd analogues has yielded
`compounds with enhanced activity/toxicity profiles [15,16]. These
`analogues are currently being characterized and have been shown
`to segregate into at least two distinct classes. The SelCIDs™
`(selective cytokine inhibitory drugs) consist of phosphodiesterase
`type 4 (PDE4) inhibitors [17]. The IMiDs™ (immunomodulatory
`drugs) are thought to be mechanistically similar to Thd, although
`with enhanced potency and therapeutic indices, and act via as yet
`75
`
`IPR2018-01714
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`
`

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`76
`
`J. B. Marriott
`
` et al.
`
`unknown mechanism(s) [18–24]. Both groups of compounds
`α
`,
`are potent inhibitors of monocyte/macrophage-derived TNF-
`although T cell co-stimulatory activity is limited to the IMiD
`group [17].
`Thd and its co-stimulatory IMiD analogues, in particular CC-
`4047 and CC-5013, are currently being assessed in the treatment
`of patients with advanced multiple myeloma and patients with
`advanced solid tumours [25–32]. Furthermore, we have shown
`recently that CC-4047, when co-administered with autologous or
`allogeneic tumour cell vaccination, is able to enhance protective
`and long-lasting immunity significantly in a murine model of col-
`orectal cancer [33].
`α
` during
`Thd and its derivatives are known to suppress TNF-
`the activation of inflammatory pathways by LPS, but it is not
`known whether this is the case during the direct stimulation of T
`cell activation pathways. Therefore, in this study we planned to
`determine the effect of these compounds, plus the non-co-
`α
` and its
`stimulatory analogue CC-3052, on the expression of TNF-
`. Furthermore, we wanted
`receptors during co-stimulation
`in vitro
`+
`+
` and CD8
` populations
`to assess the co-stimulatory effect on CD4
`+
` T cells were acti-
`because previous results suggested that CD8
`vated preferentially. Because signals generated via the TNF sys-
`tem are important in T cell homeostatic control we hoped to
`provide insights into the T cell co-stimulatory mechanism of these
`novel compounds. Finally, we hoped to show that any effects on
`the TNF system were relevant for clinical non-inflammatory dis-
`ease and to this end we obtained sera from a small phase I study
`of CC-5013 in the treatment of advanced cancer.
`
`MATERIALS AND METHODS
`
`PBMC and whole blood culture
`Thd, two IMiDs (CC-4047 and CC-5013) and a SelCID (CC-3052)
`(provided by Celgene Corporation, NJ, USA) were dissolved
`freshly in DMSO to make a 20 mg/ml stock solution. The com-
`pounds were then diluted directly into the tissue culture wells at
`the required concentrations.
`Venous blood was collected into sodium heparin vacutainers
`(Becton Dickinson, Oxford, UK) and PBMC were prepared sub-
`sequently by density centrifugation over Ficoll Hypaque (Sigma,
`g
` for 20 min cells were
`Poole, UK). After centrifugation at 700
`collected from the interface, washed three times in HBSS
`×
`6
` 10
`/ml in complete
`(Sigma), counted and resuspended at 1–2
`RPMI-1640 medium supplemented with 10% fetal calf serum
` L-glutamine (all
`(FCS), 1% penicillin/streptomycin and 2 m
`M
`±
`−
`µ
` Thd/analogues (1 ng
`10
`g/ml;
`Sigma). PBMC were cultured
`0·05%v/v final DMSO concentration) at 1 ml/well in 24-well
`µ
`g/ml; R&D systems,
`plates, precoated with anti-CD3 mAb (2·5
`Minneapolis, USA) in bicarbonate coating buffer pH 9·5. PBMC
`−
`°
`7 days at 37
`C in 5% CO
`. For some
`were incubated for 12 h
`2
`experiments PBMC were co-incubated with neutralizing anti-IL-
`µ
`g/ml; R&D systems). Cell-free supernatants
`2 mAb (0·005–0·1
`−
`°
`70
`C until assayed by
`were collected and stored in aliquots at
`ELISA. Cells were harvested and resuspended in PBS/BSA prior
`to processing as described below.
` serotype
`For lipopolysaccharide (LPS; Sigma,
`Escherichia coli
`0127:B8)-stimulated cultures heparinized venous blood was
`diluted 1 : 4 in complete RPMI-1640 medium (without FCS) and
`µ
`g/ml). All
`stimulated in 24-well plates at 1 ml/well with LPS (1
`°
`±
`C in 5% CO
`
` Thd/analogues
`cultures were incubated at 37
`2
`µ
`g/ml). After 24–48 h cell-free supernatants were collected by
`(10
`
`microcentrifugation and stored in aliquots at
`by ELISA.
`
`−
`
`°
`
`70
`
`C until assayed
`
`Clinical CC-5013 study: patient selection, treatment and sample
`collection
`We collected serum from a small phase I study designed to assess
`the safety, tolerability and efficacy of REVIMID™ (CC-5013) in
`the treatment of patients with advanced and heavily pretreated
`cancer. Of the 20 patients enrolled we obtained samples from 11
`subjects, aged between 18 and 75 years, with histologically proven
`=
`
` 6), histological or cytological
`stage IV metastatic melanoma (
`n
`=
`
` 2) or
`proof of adenocarcinoma of the exocrine pancreas (
`n
`=
`
`other malignancy (one each of breast, renal and lung cancer;
`n
`3). Before initiating therapy, patients were subject to a complete
`medical history, physical examination and baseline evaluation of
`signs and symptoms, including full blood counts and neurological
`examination. These were repeated at weekly intervals during the
`study period and also at the end of the study.
`All patients gave written informed consent prior to participa-
`tion in the study. The protocol was approved by the Wandsworth
`Local Research Ethics Committee (LREC). All patients were
`treated with 5 mg oral REVIMID™ daily (tablets were taken
`every evening) for 1 week with the dose escalating to 10 mg daily
`at week 2, 25 mg daily at week 3 and 50 mg daily at week at
`which the treatment was maintained indefinitely depending on
`tolerance.
`Blood was taken at the same time at each visit (and therefore
`at the same time relative to drug administration), collected into
`serum separator (SST; Vacutainer, BD) tubes and left to clot for
`∼
`g
`30 min. Tubes were spun at 950
` for 10 min and serum collected.
`−
`°
`70
`C until being assayed for TNF-
`Sera were frozen in aliquots at
`α
`α
` and sIL-2 receptor
` (sIL-2R) by ELISA. Serum was collected
`at baseline, during the first 1–3 weeks (10–25 mg daily) of treat-
`ment and again at weeks 4–5 (50 mg daily).
`REVIMID™ tablets were manufactured by Penn pharmaceu-
`ticals (Tredegar, Wales, UK) and supplied by Celgene Corpora-
`tion (Warren, NJ, USA).
`
`Enzyme-linked immunosorbent assay (ELISA)
`Culture supernatants were assayed for sTNFR1, sTNFR2, sIL-2R
`using kits provided by R&D Systems (Minneapolis, MN, USA)
`α
` using an assay procedure and reagents
`and for IL-2 and TNF-
`provided by Pharmingen (BD). Briefly, 96-well flat-bottomed
`°
`C with anti-
`ELISA plates (Nunc) were precoated overnight at 4
`cytokine capture mAb in bicarbonate coating buffer, pH 9·5 and
`) prior to incubation of standards and
`washed (with PBS/Tween
`20
`°
`C. After washing, plates
`test (supernatant) samples overnight at 4
`were incubated with biotinylated anticytokine mAb for 1 h. After
`further washing, plates were developed using chromogen (tetram-
`ethylbenzidine)/hydrogen peroxide for 20–30 min at RT. Stan-
`dard absorbances (405 nm) of duplicate wells (minus control zero
`standard) were used to calculate concentration of cytokine/recep-
`tor levels which were corrected for any dilution factor. Data are
`expressed as percentage compared to control (DMSO alone),
`which is represented as 100%.
`α
`, sTNFR2, IL-2
`Sera from patients were assayed for TNF-
`and sIL-2R using kits provided by R&D Systems. In each case,
`the manufacturer’s instructions were followed exactly. Data are
`expressed as pg/ml at baseline (pretreatment) compared to fol-
`low-up at 1–3 and 4–5 weeks.
`
`© 2002 Blackwell Publishing Ltd,
`
`
`
`Clinical and Experimental Immunology,
`
`
`
`130:75–84
`
`IPR2018-01714
`Celgene Ex. 2009, Page 2
`
`

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`
`a
`Diverse effects of Thd and its analogues on TNF-
`/TNFR2 during T cell co-stimulation
`
`77
`
`Flow cytometric analysis of surface CD25, CD69, TNFR1 and
`TNFR2
`PBMC were harvested from 24-well plates by gentle aspiration,
`washed and suspended in wash buffer (PBS/0·5% BSA/0·1%
`α
`CD3-
`sodium azide). Three-colour phenotypical analysis of
`stimulated PBMC was performed by surface staining cells (by
`°
`C) with the following fluoro-
`incubating cells for 30 min at 4
`chrome conjugated monoclonal antibodies: anti-CD25 FITC
`(Becton Dickinson Immunocytometry Systems, BDIS, Oxford,
`UK), anti-CD69 FITC (L78; BDIS) or anti-TNFR1 FITC
`(16803·161; R&D Systems) and anti-TNFR2 PE (22235·311;
`R&D Systems) with either anti-CD4 PerCP (SK3; BDIS) or anti-
`CD8 PerCP (SK1; BDIS) plus appropriate isotype-matched and
`compensation controls. Cells were then washed once in 2 ml wash
`µ
`l Cellfix™ (BDIS) prior to
`buffer and fixed for analysis in 200
`flow cytometric analysis. Upon analysis lymphocytes were gated
` side scatter (SSC) properties.
`on forward scatter (FSC)
`versus
`PerCP-positive T cell subsets were then selected by gating on SSC
` FL-3 dot-plots and displayed as two-colour dot-plots, with
`versus
`quadrants set according to isotype-matched controls. For each
`sample 10 000 lymphocytes were acquired on a Becton Dickinson
`FACScan using CellQuest™ software.
`
`α
`Analysis of intracellular TNFR2, IL-2 and TNF-
`α
`±
`PBMC were stimulated with
`CD3
` CC-4047 for 24–96 h with
`µ
`g/ml; Sigma)
`the protein transport inhibitor Brefeldin A (10
`present for the last 6 h of culture to allow intracellular accumu-
`lation of protein. Cells were harvested, washed and suspended in
`wash buffer, then fixed and permeabilized using the specially for-
`mulated Becton Dickinson FACS lysis and permeabilization solu-
`tions as per the manufacturer’s instructions. Cells were then
`stained with anti-TNFR2 FITC, anti-IL-2 FITC (5344·111, BDIS)
`α
` FITC (6401·1111, BDIS) and anti-CD4 PE or anti-
`or anti-TNF-
`CD8 PE plus appropriate isotype-matched and compensation
`controls for 30 min before washing once in 2 ml wash buffer fol-
`µ
`l Cellfix™. Stained samples
`lowed by a final resuspension in 200
`°
`C prior to two-colour analysis on the
`were kept in the dark at 4
`permeabilized cells. Upon flow cytometric analysis lymphocytes
` side scatter (SSC)
`were gated on forward scatter (FSC)
`versus
`properties and displayed as two-colour dot-plots, with quadrants
`set according to isotype-matched controls. For each sample 10 000
`lymphocytes were acquired on a Becton Dickinson FACScan
`using CellQuest™ software.
`
`Proliferation assay
`×
`5
`PBMC (1–2
` 10
`) were resuspended in complete RPMI-1640
`±
`α
` Thd/analogues) onto
`CD3 precoated 96-well
`round-
`(
`µ
`l per well.
`bottomed plates (Falcon, BD) to a final volume of 200
`°
`3
`C in 5% CO
` for 5 days with [
`H]-
`Cells were cultured at 37
`2
`thymidine (NEN Life Science Products, Hounslow, UK) added
`for the last 24 h (2 mCi/well). Cells were harvested onto glass fibre
`filter paper and counted in a matrix 96 direct beta counter (Can-
`3
`H]-thymidine was determined as
`berra Packard). Uptake of [
`mean cpm of sextuplet culture wells and expressed as percentage
`change compared to control (DMSO alone).
`
`Statistical analysis
`For analysis of serum data comparisons between groups were
`examined by the Kruskal–Wallis test with inclusion of the Dunn’s
`
`multiple comparison test. The statistics were performed using
`Primer of Biostatistics (3·02) statistical software.
`
`RESULTS
`
`Co-stimulatory IMiD CC-4047 strongly inhibits the secretion
`α
`of sTNFR2 by
`CD3-stimulated PBMC cultures whereas
`non-co-stimulatory SelCID CC-3052 augments sTNFR2
`A single treatment of CC-4047 leads to potent inhibition of
`α
`CD3-stimulated PBMC supernatants at 24 h
`sTNFR2 levels in
`(Fig. 1a). This effect was consistent in four of four experiments
`α
`±
`=
`CD3 alone, 2676 pg/ml; + CC-4047
` 32·3%
`(24 h: mean
`23·5% of control). Inhibition was decreased at 48 h (mean
`α
`±
`=
`CD3 alone, 4436 pg/ml; + CC-4047
` 52·7%
` 13% of control),
`although a small inhibitory effect was still apparent after 72 h.
`Soluble TNFR2 was not consistently detectable in 12 h cultures
`(data not shown).
`We found that the non-co-stimulatory analogue, CC-3052,
`had the opposite effect on sTNFR2 when compared to CC-4047 in
`±
`=
` 222%
` 141% of control). The
`all experiments (24 h: + CC-3052
`±
`=
` 159%
`effect of CC-3052 also decreased at 48 h (+ CC-3052
`50·4%) and a smaller augmentary effect was apparent at 72 h.
`Thd had very little overall effect on sTNFR2 (for example, at
`±
`=
` 102·7%
` 9·3% of control).
`48 h: + Thd
`In contrast to the effects on sTNFR2 none of the compounds
`had a consistent or significant effect on the levels of sTNFR1 at
`±
`±
` 10·9%; CC-3052, mean 86·8%
`24 h (+ CC-4047, mean 110%
`±
`=
`13·6%; Thd
` 104%
` 11·2%; Fig. 1b). However, CC-3052 did
`inhibit sTNFR1 at 48 and 72 h.
`We also found that CC-4047 and Thd strongly induced the
`α
`α
` at 72 h (mean
`CD3 alone control, 1443 pg/
`production of TNF-
`±
`±
`=
`=
` 365%
` 233%; + Thd
` 247%
` 122%; Fig. 1c),
`ml; + CC-4047
`α
`CD3, 2753 pg/ml;
`although there was very little effect at 24 h (
`±
`±
`=
`=
` 114%
` 37·8%; Thd
` 129%
` 39·8%).
`CC-4047
`
`sTNFR2 production by LPS-stimulated whole blood cultures
`was not affected by CC-4047, Thd or CC-3052
`We next determined whether the divergent effects of CC-4047
`and CC-3052 on sTNFR2 production seen during T cell co-
`α
`CD3 were also apparent during LPS activation
`stimulation with
`of monocyte/macrophage cell populations. In contrast to the
`effect during T cell activation we found that there was no effect of
`any compound on sTNFR1 or sTNFR2 production by LPS-
`stimulated whole blood cultures at any time-point (Fig. 1d,e).
`As expected, CC-3052 and CC-4047 both strongly inhibited LPS-
`α
` production (Fig. 1f) and were more effective than
`induced TNF-
`Thd in this respect.
`
`CC-4047 and CC-5013 strongly reduced the surface expression
`α
`+
`+
`of TNFR2 on
`CD3-stimulated CD4
` and CD8
` T cells during
`PBMC co-stimulation
`We then determined whether the inhibitory effect of CC-4047 on
`supernatant levels of sTNFR2 during co-stimulation was due to
`reduced surface TNFR2 expression or to reduced receptor shed-
`ding. We found that CC-4047 and CC-5013 (and to a lesser extent
`Thd) reduced the surface expression of TNFR2 and that this
`correlated with the induction of surface CD25 (IL-2 receptor)
`+
` (Fig. 2a)
`(Fig. 2). Furthermore, this was seen equally in both CD4
`+
` populations (Fig. 2b). Results from two dose–response
`and CD8
`experiments indicated that co-stimulation (gauged by CD69
`
`© 2002 Blackwell Publishing Ltd,
`
`
`
`Clinical and Experimental Immunology,
`
`
`
`130:75–84
`
`IPR2018-01714
`Celgene Ex. 2009, Page 3
`
`

`

`
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`
`
`24
`
`48
`h
`
`72
`
`96
`
`24
`
`48
`
`72
`
`h
`
`J. B. Marriott et al.
`
`(c)
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`0
`
`(f)
`
`% TNF-alpha relative to control
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`0
`
`% TNF-alpha relative to control
`
`24
`
`48
`h
`
`72
`
`96
`
`24
`
`48
`
`72
`
`h
`
`(b)
`
`500
`
`400
`
`300
`
`200
`
`100
`
`% TNFR1 relative to control
`
`0
`
`0
`
`(e)
`
`500
`
`400
`
`300
`
`200
`
`100
`
`% TNFR1 relative to control
`
`0
`
`0
`
`24
`
`48
`h
`
`72
`
`96
`
`24
`
`48
`
`72
`
`h
`
`(a)
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`0
`
`(d)
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`0
`
`78
`
`% TNFR2 relative to control
`
`% TNFR2 relative to control
`
`Fig. 1. Co-stimulatory CC-4047 strongly inhibits the secretion of sTNFR2 by αCD3-stimulated PBMC cultures (but not LPS-stimulated
`cultures) whereas non-co-stimulatory SelCID CC-3052 augments sTNFR2. Divergent effects of CC-4047 (Ο), CC-3052 (䊐), and Thd (∆)
`on the production of sTNFR2, sTNFR1 & TNF-α by PBMC-stimulated with αCD3 (a–c) and whole blood cultures stimulated with LPS
`(d–f). Cultures were incubated ± Thd/analogues (10 µg/ml) and supernatants collected at the indicated times as per Materials and methods.
`Results are expressed as percentage change compared to control cultures with DMSO alone. Data presented are from four normal donors
`used in two separate experiments.
`
`expression) and inhibition of surface TNFR2 could both be
`detected at CC-4047 concentrations as low as 10 ng (data not
`shown).
`We were unable to detect the presence of surface TNFR1
`either with αCD3 alone or in the presence of the co-stimulatory
`compounds using cells from a total of four donors in two separate
`experiments (data not shown).
`
`CC-4047 did not alter the level of total cellular TNFR2
`(intracellular plus surface) in T cells during PBMC co-
`stimulation
`We investigated whether the inhibitory effect of CC-4047 on sur-
`face TNFR2 was due to the inhibition of TNFR2 protein produc-
`tion. We utilized the protein transport inhibitor Brefeldin A and
`two-colour flow cytometry in order to measure the total amount
`of cellular TNFR2 within CD4+ and CD8+ cells during PBMC co-
`stimulation. The data presented in Fig. 3 show that when total
`protein was analysed CC-4047 did not appear to exert an inhibi-
`tory effect on TNFR2. The data presented suggest strongly that
`TNFR2 protein production itself is not being affected. Therefore,
`it is likely that trafficking to the cell surface that is being inhibited.
`Furthermore, our data also rule out an inhibitory effect on
`TNFR2 transcription.
`
`The relative extent of TNFR2 inhibition by CC-4047, CC-5013
`and Thd corresponds to co-stimulatory activity as gauged by
`the production of IL-2, sIL-2R and proliferation in αCD3
`stimulated PBMC cultures
`We then investigated whether there was a possible connection
`between the inhibitory effect of these compounds on TNFR2 sur-
`face expression and co-stimulatory activity. The co-stimulatory
`activity of CC-4047, CC-5013 and Thd was assessed by IL-2 and
`sIL-2R production and also by the incorporation of tritiated thy-
`midine (to measure proliferation).
`We found that IL-2 production was not detected in control
`(αCD3 alone) cultures. However, after 24 h CC-4047, CC-5013
`and, to a lesser extent Thd, strongly induced IL-2 production
`(Fig. 4a). For CC-4047 this effect was still apparent after 7 days of
`culture. The increased expression of sIL-2R became apparent
`after 72 h and followed a very similar pattern (Fig. 4b). Prolifer-
`ation was assessed at days 5–6 with a consistent pattern of results
`(Fig. 4c). Therefore, using these three markers of co-stimulation
`the same relative pattern of effectiveness emerged: CC-
`4047 > CC-5013 >> Thd.
`In contrast to these effects the non-co-stimulatory SelCID,
`CC-3052, strongly inhibited IL-2 and sIL-2R although it had no
`effect on proliferation, as we have shown previously [19].
`
`© 2002 Blackwell Publishing Ltd, Clinical and Experimental Immunology, 130:75–84
`
`IPR2018-01714
`Celgene Ex. 2009, Page 4
`
`

`

`Diverse effects of Thd and its analogues on TNF-a/TNFR2 during T cell co-stimulation
`
`79
`
`(a)
`
`a CD3 alone
`
`a CD3 + CC-4047
`
`a CD3 + CC-5013
`
`a CD3 + Thd
`
`100101102103104
`
`100 101 102 103 104
`CD25 FITC
`
`TNF-R2 PE
`
`100 101 102 103 104
`CD25 FITC
`
`100101102103104
`
`TNF-R2 PE
`
`100 101 102 103 104
`CD25 FITC
`
`100101102103104
`
`TNF-R2 PE
`
`100 101 102 103 104
`CD25 FITC
`
`100101102103104
`
`48·81
`
`17·38
`
`22·28
`
`46·33
`
`100101102 103 104
`TNF-R2 PE
`
`100101102 103 104
`TNF-R2 PE
`
`100101102 103 104
`TNF-R2 PE
`
`100101102 103 104
`TNF-R2 PE
`
`114·03
`
`250·22
`
`210·49
`
`165·96
`
`Events
`
`Events
`
`100101102 103 104
`CD25 FITC
`
`100101102 103 104
`CD25 FITC
`
`100101102 103 104
`CD25 FITC
`
`100101102 103 104
`CD25 FITC
`
`(b)
`
`a CD3 alone
`
`a CD3 + CC-4047
`
`a CD3 + CC-5013
`
`a CD3 + Thd
`
`100101102103104
`
`100101102103104
`
`100 101 102 103 104
`CD25 FITC
`
`TNF-R2 PE
`
`100 101 102 103 104
`CD25 FITC
`
`TNF-R2 PE
`
`100 101 102 103 104
`CD25 FITC
`
`100101102103104
`
`TNF-R2 PE
`
`100 101 102 103 104
`CD25 FITC
`
`100101102103104
`
`TNF-R2 PE
`
`TNF-R2 PE
`
`41·18
`
`10·73
`
`14·97
`
`35·84
`
`100101102 103 104
`TNF-R2 PE
`
`100101102 103 104
`TNF-R2 PE
`
`100101102 103 104
`TNF-R2 PE
`
`100101102 103 104
`TNF-R2 PE
`
`99·20
`
`149·64
`
`149·19
`
`145·49
`
`Events
`
`Events
`
`100101102 103 104
`CD25 FITC
`
`100101102 103 104
`CD25 FITC
`
`100101102 103 104
`CD25 FITC
`
`100101102 103 104
`CD25 FITC
`
`Fig. 2. CC-4047 and CC-5013 strongly reduced the surface expression of TNFR2 on αCD3 stimulated CD4+ and CD8+ T cells during PBMC
`co-stimulation. PBMC were incubated ± Thd/analogues (10 µg/ml) for 48 h. Three-colour flow cytometric analysis was performed by surface
`staining stimulated PBMC with anti-CD25 FITC, anti-TNFR2 PE and anti-CD4/CD8 PerCP plus appropriate isotype matched and
`compensation controls as described in Materials and methods. (a) Dot plots represent TNFR2 versus CD25 (IL-2 receptor) gated on (a)
`CD4 and (b) CD8 T cell subsets. Histograms show mean fluorescent intensity of TNFR2 expression on CD25+ T cells. This effect was highly
`consistent when performed in 10 separate experiments each using PBMC from two normal donors although data from a single represen-
`tative experiment are shown.
`© 2002 Blackwell Publishing Ltd, Clinical and Experimental Immunology, 130:75–84
`
`IPR2018-01714
`Celgene Ex. 2009, Page 5
`
`

`

`80
`
`J. B. Marriott et al.
`
`Isotype matched
`control
`
`Unstimulated
`
`a CD3 alone
`
`a CD3 + CC-4047
`
`CD4 PE
`
`CD4 PE
`
`CD4 PE
`
`IgG1 FITC
`
`TNF-R2 FITC
`
`TNF-R2 FITC
`
`TNF-R2 FITC
`
`CD8 PE
`
`CD8 PE
`
`CD8 PE
`
`CD4 PE
`
`CD4
`
`CD8 PE
`
`CD8
`
`IgG1 FITC
`
`TNF-R2 FITC
`
`TNF-R2 FITC
`
`TNF-R2 FITC
`
`Fig. 3. CC-4047 did not alter the level of total cellular TNFR2 (intracellular plus surface) in T cells during PBMC co-stimulation.
`Unstimulated PBMC and cells stimulated with αCD3 ± CC-4047 (10 µg/ml) for 24 h (TNFR2) (the last 6 h in the presence of the protein
`transport inhibitor, Brefeldin A) were harvested and processed as described in Materials and methods. Lymphocytes were gated on forward
`scatter (FSC) versus side scatter (SSC) properties and displayed as two-colour dot-plots, with quadrants set according to isotype-matched
`controls. No difference was seen in the expression of intracellular TNFR2 during the co-stimulation of CD4+ or CD8+ T cells. The data
`presented are representative of three separate experiments in which very similar results were obtained.
`
`(b)
`
`(c)
`
`CC-
`3052
`
`CC-
`4047
`
`CC-
`5013
`
`Thd
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`% proliferation relative to control
`
`CC-
`3052
`
`CC-
`4047
`
`CC-
`5013
`
`Thd
`
`400
`
`300
`
`200
`
`100
`
`0
`
`% sIL-2R (at 72 h) relative to control
`
`CC-
`3052
`
`CC-
`4047
`
`CC-
`5013
`
`Thd
`
`(a)
`
`900
`
`800
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`% IL-2 (at 24 h) relative to control
`
`Fig. 4. The relative extent of TNFR2 inhibition by CC-4047, CC-5013 and Thd corresponds to the degree of co-stimulation by these
`compounds. PBMC cultures were co-incubated for 24 h (IL-2), 72 h (sIL-2R) and 120 h (proliferation) ± Thd/analogues (10 µg/ml) as
`described in Materials and methods. Cell culture supernatants were assayed for IL-2 and sIL-2R by ELISA. Proliferation was assessed by
`[3H]-thymidine incorporation. Results are expressed as percentage change in (a) IL-2, (b) sIL-2R and (c) proliferation compared to control
`cultures. Data are from four normal donors used in two separate experiments.
`
`Co-stimulation by CC-4047 and subsequent TNF-α production,
`but not early inhibition of TNFR2, is dependent on IL-2-
`mediated signalling
`We next determined the importance of IL-2-mediated signalling
`for the process of CC-4047-mediated co-stimulation. We wanted
`to determine whether elevation of TNF-α was a consequence of T
`cell activation and not a direct effect of CC-4047. We also wanted
`to confirm that TNFR2 inhibition was not due to co-stimulation
`itself and raise the possibility that this early effect may be signif-
`icant in the initiation of the co-stimulatory process.
`Inhibition of IL-2 signalling was achieved with the use of a
`neutralizing anti-IL-2 mAb precoated onto the culture well.
`Under co-stimulatory conditions anti-IL2 mAb decreased super-
`natant IL-2 levels in a dose-dependent manner (Fig. 5a) and also
`
`decreased the surface expression of IL-2 receptor (data not
`shown). In these same cultures inhibition of IL-2 prevented the
`superinduction of TNF-α in a similar dose–response fashion
`(Fig. 5b) Therefore, co-stimulation-induced TNF-α production is
`clearly mediated via IL-2 generated signalling. However, in iden-
`tical studies we found that the inhibition of IL-2-mediated signal-
`ling did not prevent TNFR2 inhibition by CC-4047 (data not
`shown).
`
`Co-stimulation-induced IL-2 and TNF-α is derived from both
`CD4+ and CD8+ T cells
`Having established a requirement for IL-2 production and (sub-
`sequent IL-2-mediated signalling) during CC-4047- mediated
`co-stimulation we sought to determine the source of this
`
`© 2002 Blackwell Publishing Ltd, Clinical and Experimental Immunology, 130:75–84
`
`IPR2018-01714
`Celgene Ex. 2009, Page 6
`
`

`

`Diverse effects of Thd and its analogues on TNF-a/TNFR2 during T cell co-stimulation
`
`81
`
`+
`–
`
`+
`+
`+
`+
`0·1 0·05 0·01 0·005
`
`(b)
`
`100
`
`80
`
`60
`
`40
`
`20
`
`co-stimulated cultures
`% TNF-alpha relative to
`
`0
`CC-4047
`+ anti-IL-2 mAb
`
`(a)
`
`100
`
`80
`
`60
`
`40
`
`20
`
`co-stimulated cultures
`
`% IL-2 relative to
`
`0
`CC-4047
`+ anti-IL-2 mAb
`
`+
`–
`
`+
`+
`+
`+
`0·1 0·05 0·01 0·005
`
`Fig. 5. Co-stimulation by CC-4047 and subsequent TNF-α production, but not early inhibition of TNFR2, is dependent on IL-2-mediated
`signalling. PBMC cultures were stimulated with αCD3 in the presence of CC-4047 (10 µg/ml) and anti-IL-2 mAb (0·1–0·005 µg/ml) as
`described in Materials and methods. Results are expressed as percentage values relative to cultures co-stimulated with αCD3 + CC-4047
`(0% represents αCD3 alone). Cultures with increasing concentrations of anti-IL-2 mAb show a reduction in (a) supernatant IL-2 levels
`and (b) supernatant TNF-α levels. Data shown are from four normal donors in two separate experiments.
`
`Unstimulated
`
`0·61%
`
`a CD3 alone
`2·74%
`
`a CD3 + CC-4047
`8·72%
`
`CD4 PE
`
`8·04%
`
`IL-2 FITC
`
`IL-2 FITC
`
`CD4 PE
`
`1·43%
`
`IL-2 FITC
`
`IL-2 FITC
`
`CD4 PE
`
`0·81%
`
`CD8 PE
`
`CD8 PE
`
`CD8 PE
`
`Unstimulated
`
`0·71%
`
`a CD3 alone
`1·57%
`
`a CD3 + CC-4047
`14·86%
`
`CD4 PE
`36·34%
`
`TNF-a FITC
`
`TNF-a FITC
`
`CD4 PE
`6·22%
`
`TNF-a FITC
`
`TNF-a FITC
`
`CD4 PE
`1·32%
`
`(a)
`
`IL-2 FITC
`
`CD4
`
`IL-2 FITC
`
`CD8
`
`(b)
`
`TNF-a FITC
`
`TNF-a FITC
`
`CD4
`
`CD8
`
`CD8 PE
`
`CD8 PE
`
`CD8 PE
`
`Fig. 6. Co-stimulation-induced IL-2 and TNF-α are derived from both CD4+ and CD8+ T cells. Unstimulated PBMC and cells stimulated
`with αCD3 ± CC-4047 (10 µg/ml) for 96 h (IL-2/TNF-α) (the last 6 h in the presence of the protein transport inhibitor, Brefeldin A) were
`harvested and processed as described in Materials and methods. Lymphocytes were gated on forward scatter (FSC) versus side scatter
`(SSC) properties and displayed as two-colour dot-plots, with quadrants set according to isotype-matched controls. The induction of
`intracellular IL-2 and TNF-α was detected in both CD4+ and CD8+ T cells. Data shown are from one normal donor and are representative
`of very similar data obtained from three donors.
`
`© 2002 Blackwell Publishing Ltd, Clinical and Experimental Immunology, 130:75–84
`
`IPR2018-01714
`Celgene Ex. 2009, Page 7
`
`

`

`82
`
`J. B. Marriott et al.
`
`(b)
`
`6000
`
`4000
`
`2000
`
`TNF-alpha (ng/mL)
`
`(a)
`
`2400
`
`2000
`
`1600
`
`1200
`
`800
`
`400
`
`sIL-2R (pg/mL)
`
`0
`Baseline
`
`Weeks 1–3 Weeks 4–5
`
`0
`Baseline Weeks 1–3 Weeks 4–5
`
`Fig. 7. Treatment of advanced cancer patients with CC-5013/REVIMID™ results in increased TNF-α and sIL-2 receptor levels. Patient
`serum samples were obtained from a small phase I study as detailed in Materials and methods. Serum was collected at baseline and twice
`after commencing CC-5013 treatment (weeks 1–3 and weeks 4–5) and assayed for (a) sIL-2R and (b) TNF-α by ELISA. Each symbol
`represents a single patient.
`
`cytokine. In particular we wished to determine conclusively
`whether co-stimulation affects both CD4+ and CD8+ T cells. This
`was achieved by intracellular cytokine specific staining of CD4+
`and CD8+ T cells within the PBMC population. Data are shown
`from one of two separate experiments in which almost identical
`data were obtained. Figure 6a confirms the lack of IL-2 produc-
`tion observed by ELISA in control (αCD3 alone) cultures
`because very few cells from either subset produce IL-2. However,
`upon CC-4047-mediated co-stimulation there are increased num-
`bers of IL-2-producing cells from both T cell subsets. Further-
`more, the elevated production of TNF-α, which is secondary to
`co-stimulation, is also due to both T cell types (Fig. 6b). Thus,
`co-stimulation by CC-4047 affects CD4+ and CD8+ T cell subsets
`rather than inducing preferential activation of CD8+ cells, as has
`been reported previously [8].
`
`Treatment of cancer patients with CC-5013/REVIMID™ leads
`to increased serum levels of sIL-2R and TNF-α
`We wanted to extend our in vitro observations and determine
`whether the clinical use of a co-stimulatory IMiD (CC-5013/
`REVIMID™) could lead to elevation of serum TNF-α in the c