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`ofAugust 18, 2018.
`
`Differential Cytokine Modulation and T Cell
`meJournal of
`
`DB Immunology Activation by Two Distinct Classes of
`Thalidomide Analogues That Are Potent
`Inhibitors of TNF- o
`Laura G. Corral, Patrick A. J. Haslett, George W. Muller,
`Roger Chen, Lu-Min Wong, ChristopherJ. Ocampo,
`Rebecca T. Patterson, David I. Stirling and Gilla Kaplan
`JImmunol 1999; 163:380-386;:
`http://www.jimmunol.org/content/163/1/380
`
`eee
`
`References
`
`This article cites 44 articles, 11 of which you can accessforfree at:
`http://www.jimmunol.org/content/163/1/3 80.full#ref-list- 1
`
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`The JournalofImmunology is published twice each month by
`The American Association of Immunologists, Inc.,
`1451 Rockville Pike, Suite 650, Rockville, MD 20852
`Copyright © 1999 by The American Association of
`Immunologists All rights reserved.
`Print ISSN: 0022-1767 Online ISSN: 1550-6606.
`
`
`
`ALVOGEN, Exh. 1009, p. 0001
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`810%‘81IsNSNYyUoysondAq/sx0"jounUNamay/:dyyTOYpopropumog
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`ALVOGEN, Exh. 1009, p. 0001
`
`

`

`Differential Cytokine Modulation and T Cell Activation by
`Two Distinct Classes of Thalidomide Analogues That Are
`Potent Inhibitors of TNF-a!
`
`Laura G. Corral,”* Patrick A. J. Haslett,' George W. Muller,* Roger Chen,* Lu-Min Wong,*
`Christopher J. Ocampo,’ Rebecca T. Patterson,* David I. Stirling,* and Gilla Kaplan‘
`TNF-a mediates both protective and detrimental manifestations of the host immune response. Our previous work has shown
`thalidomideto be a relatively selective inhibitor ofTNF-a production in vivo andin vitro. Additionally, we have recently reported
`that thalidomide exerts a costimulatory effect on T cell responses. To develop thalidomide analogues with increased anti-TNF-a
`activity and reduced or absenttoxicities, novel TNF-a inhibitors were designed and synthesized. When a selected group of these
`compounds was examined for their immunomodulatory activities, different patterns of cytokine modulation were revealed. The
`tested compoundssegregated into twodistinct classes: one class of compounds, shownto be potent phosphodiesterase 4 inhibitors,
`inhibited TNF-a production, increased IL-10 production by LPS-induced PBMC,and hadlittle effect on T cell activation; the
`other class of compounds, similar to thalidomide, were not phosphodiesterase 4 inhibitors and markedly stimulated T cell pro-
`liferation and IL-2 and IFN-y production. These compounds inhibited TNF-a, IL-1, and IL-6 and greatly increased IL-10
`production by LPS-induced PBMC.Similarto thalidomide, the effect of these agents on IL-12 production was dichotomous; IL-12
`was inhibited when PBMC werestimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The
`latter effect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatoryactivities of these classes
`of thalidomide analogues may potentially allow them to be used in the clinic for the treatment ofdifferent immunopathological
`disorders. The Journal ofImmunology, 1999, 163: 380-386.
`
`umornecrosis factor a, a highly pleiotropic cytokine pro-
`duced primarily by monocytes and macrophages, plays a
`central role in the host protective immune response to
`bacterial and viral infections. For example, TNF-a is essential for
`granuloma formation and the control ofbacterial dissemination in
`experimental tuberculosis in mice(1, 2). In addition, TNF-a added
`to infected cells in vitro inhibits the replication of both DNA and
`RNAviruses (3, 4). However, the cytokine may also play a role in
`the pathogenesis of disease. Perhaps the best evidence for this is
`the dramatic reduction in disease activity observed in rheumatoid
`arthritis and inflammatory boweldisease after treatment ofpatients
`with neutralizing anti-TNF-a Abs (5, 6). Additionally, elevated
`levels of TNF-a have been associated with the fevers, malaise, and
`weight loss that accompany chronic infections (7), and reductions
`in TNF-a levels have beenlinked with an amelioration of clinical
`symptoms in a numberof disease states (8-11).
`Our previous work has shown thalidomide to be a relatively
`selective inhibitor of TNF-a production by human monocytes in
`vivo and in vitro. Leprosy patients with erythema nodosum lepro-
`sum treated with thalidomide, experience a reduction of serum
`TNF-« levels with a concomitant abrogation ofclinical symptoms
`
`
`*Celgene Corporation, Warren, NJ 07059; and ‘Laboratory of Cellular Physiology
`and Immunology, The Rockefeller University, New York, NY 10021
`Received for publication December 23, 1998. Accepted for publication April 22, 1999.
`The costs of publication ofthis article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance
`with 18 U.S.C. Section 1734 solely to indicate this fact.
`' This work was supported by Celgene Corporation. G.K was supported by Public
`Health Service National Institutes of Health Research Grant AI-22616; P.A.J.H. was
`supported by General Clinical Research Center Grant MOI-RROO102.
`? Address correspondence andreprint requests to L. G. Corral, Laboratory of Cellular
`Physiology and Immunology, Rockefeller University, 1230 York Avenue, New York,
`NY 10021. E-mail address; corrall@rockvax.rockefeller.edu
`
`Copyright © 1999 by The American Association of Immunologists
`
`(9). In patients with tuberculosis, with or without HIV infection,
`thalidomide lowers plasma TNF-a protein levels and leukocyte
`TNF-a mRNAlevels in association with an accelerated weight
`gain (8). In vitro, thalidomide has been shown to selectively par-
`tially (50-70%)inhibit TNF-a produced by monocytes and mac-
`rophagesstimulated with LPS (12).
`Recently, we have reported the ability of thalidomide to co-
`stimulate T cells in vitro (13). Thus, in addition to its monocyte
`cytokine-inhibitory activity, thalidomide exerts a costimulatory or
`adjuvant effect on T cell responses that includes increased produc-
`tion of IL-2 and IFN-y. This effect may contribute to the immune-
`modulating effects of the drug.
`To obtain drugs that are more efficient TNF-a inhibitors than
`thalidomide, structural analogues of the parent molecule have been
`synthesized and examinedforinhibition of TNF-a production. Re-
`cently, some of these thalidomide analogues have been described
`(12, 14, 15, 47). On a molar basis, these reported compoundsare
`up to 50,000-fold more potent
`than thalidomide at
`inhibiting
`TNF-a production by PBMCinvitro. In this study, we have se-
`lected six of these compounds and evaluated them for their effects
`on the production of other monocyte cytokines, as well as their
`immunomodulatory effects on T cells.
`
`Materials and Methods
`Preparation of cells
`PBMCwere isolated from the blood of healthy volunteers by Ficoll-
`Hypaque (Pharmacia, Piscataway, NJ) density centrifugation as previously
`described (12). T lymphocytes were purified from PBMCbyrosetting with
`neuraminidase-treated sheep erythrocytes and subsequent incubation of
`erythrocyte-rosetting cells on a nylon wool column. Nonadherentcells
`eluted from the column were >93% CD3 Ag positive by flow cytometry
`(FACStar, Becton Dickinson, San Jose, CA). Leukocytes were cultured in
`RPMI medium (Life Technologies, Grand Island, NY) supplemented with
`
`0022-1767/99/$02.00
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`ALVOGEN, Exh. 1009, p. 0002
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`8107‘8TsndnyuoysondKq/s10jounuunlMaay/:dnywoypeprojumoq
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`ALVOGEN, Exh. 1009, p. 0002
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`

`

`The Journal of Immunology
`
`10% AB* human serum, 2 mM L-glutamine, 100 UAnlpenicillin, and 100
`pg/ml streptomycin (Life Technologies).
`
`Thalidomide and analogues
`Thalidomide and analogues (Celgene, Warren, NJ) were dissolved in
`DMSO(Sigma, St. Louis, MO); further dilutions were made in culture
`medium immediately before use. The final DMSO concentration in all
`assays was 0.25%. The followingstructural analogues were used: CIIL-A is
`compound 3a (14) and CC-1069 (12); CII-B is compound CC-3052 (16)
`with the carboxymethyl group replaced by an amide moiety; CII-C is an
`amino-substituted analogue of compound 4b (14); compounds CI-A, CL-B,
`and CI-C are amino-substituted analogues ofthalidomide. CI-A is 5a, CB
`is 8a and CI-C is 14 (47).
`
`PBMCstimulation by LPS
`PBMC (2 X 10° cells) incubated in 96-well flat-bottom polystyrene Costar
`tissue culture plates (Corning, Corning, NY) were stimulated by 1 ug/ml
`LPS from Salmonella minnesota R595 (List Biological Labs, Campbell,
`CA)for the induction of TNF-o , IL-1, IL-6, IL-8, IL-10, and IL-12 (12).
`Cells were incubated with or without thalidomide or analogues for 20 h,
`and supernatants werecollected for the determination of cytokinelevels by
`ELISA.
`
`PBMCstimulation by anti-CD3 Ab
`PBMC(1 X 10° cells) were stimulated by cross-linking of the TCR by
`immobilized monoclonal mouse anti-human CD3 (Orthoclone OKT3, a
`kind gift of Dr. R. Zivin, Orthobiotech, Raritan, NJ) as previously de-
`scribed (13). The anti-CD3 Ab wasdiluted to 10 pg/ml in 100 xl PBS and
`coated onto 48-well flat-bottom polystyrene Falcon tissue culture plates
`(Becton Dickinson, Franklyn Lakes, NJ) by overnight incubation at 4°C.
`Appropriate dilutionsof thalidomide and analogues were addedatthestart
`of the cell cultures. Supernatants were collected at 24, 48, and 72 h and
`assayed for IL-10, IL-12, and TNF-a levels. Cells were collected at 48h
`for evaluation of CD40 ligand (CD40L)3 and CD3 surface expression by
`two-color flow cytometry (anti-CD40L, PharMingen, San Diego, CA; anti-
`CD3, Becton Dickinson, San Jose, Ca).
`
`T cell stimulation andproliferation assays
`Purified T cells (2 < 10° cells/well) in 96-well Costartissue culture plates
`(Corning) previously coated with anti-CD3 mAb (as above) were treated
`daily with thalidomide or analogues for up to 120 h. Supematants were
`harvested for IFN-y assay at 72 h. T cell-proliferative responses were as-
`sayed by measuring [*H]thymidine (NEN Products, Boston, MA) incorpo-
`ration during the last 18 h of 120-h cultures. DNA was harvested onto fiber
`mats with an automatic cell harvester (Skatron, Stirling, VA), and [*H]thy-
`midine incorporation was measured with a LKB 1205 Betaplate liquid
`scintillation counter (Wallac, Gaithersburg, MD)
`
`
`
`Phosphodiesterase 4 (PDE4) inhibition assay
`PDE4inhibition was evaluated in purified extracts of promonocytic U937
`cells using a modified method of Hill and Mitchell (17) as previously
`described (14). Cells (1 X 10°) were washed in PBS and lysed in cold
`homogenization buffer (20 mM Tris-HCl, pH 7.1; 3 mM 2-ME;
`1 mM
`MgCl; 0.1 mM EGTA,
`| «M PMSF, 1 »g/ml leupeptin). After homoge-
`nization with a Dounce homogenizer, the supernatant was collected by
`centrifugation and loaded onto a Sephacryl S-200 column equilibrated with
`homogenization buffer. PDE4 was eluted in homogenization buffer, and
`enzymeactivity was determined in 50 mM Tris-HCl, pH 7.5, 5 mM MgCl,
`and 1 wM cAMP (of which 1% was [SH]cAMP)as described in detail by
`Thompsonetal. (18). Reactions were performed at 30°C for 30 min and
`terminated by boiling for 2 min. Briefly, cyclic 3’,5’-[7H]AMP was con-
`verted to 5’-GHJAMP by phosphodiesterase. The separation of 5’-
`PHIJAMPfrom 3',5’-PH]AMP wasachieved by enzymatically converting
`5'-PHJAMPto [H]adenosine with nucleotidase present in snake venom
`(Sigma, V-0376),
`1 mg/ml at 30°C for 15 min. Adenosine was separated
`from the unreacted cyclic substrate by addition of 200 pl of AGI-X8resin
`(Bio-Rad, Hercules, CA) that absorbs cyclic 3',5'-[PHJAMP. Samples were
`then spun at 3000 rpm for 5 min, and 50 pil of the aqueous phase were
`taken for counting of adenosine radioactivity by liquid scintillation tech-
`niques. Enzyme activity was determined in the presence of varying con-
`centrations of compounds. ICs) values were determined from dose-re-
`sponsescurves derived fromat least three independent experiments done in
`
`> Abbreviations used in this paper: CD40L, CD40ligand; PDE4, phosphodiesterase 4.
`
`381
`
`duplicate. ICs) values were calculated by nonlinear regression analysis
`(variable slope) using Prism by GraphPad Software (San Diego, CA).
`Cytokine assays
`
`Culture supernatants were harvested at indicated times and frozen imme-
`diately at —70°C until assayed in triplicate or duplicate. TNF-a , IL-1B,
`IL-2,IL-6, IL-8, IL-10, IL-12 (p40 and p70), and IFN-y levels were mea-
`sured by ELISA (Endogen, Cambridge, MA) as described by the
`manufacturer.
`
`Statistical analysis
`Data were evaluated by the Friedmantest, a nonparametric ANOVA,in
`view of the small sample size. The SPSS computer program was used.
`Significance was set at p < 0.05.
`
`Results
`Effect of thalidomide analogues on LPS-induced cytokine
`production
`A group of thalidomide analogues wereselectedfor their capacity
`to inhibit TNF-a production by LPS-stimulated PBMC.Their IC55
`values for TNF-« (the concentration at which each compound was
`able to inhibit TNF-a levels by 50%) were established when
`screening these agents (Table I). Althoughall compounds were
`efficient TNF-a inhibitors, their dose-response curves were not
`identical (data not shown). For some compounds, dose-response
`curves were the classical sigmoidal curves seen for pharmacolog-
`ical antagonists (class II compounds, see below) (12). Other com-
`pounds, however, showeda flatter, thalidomide—like dose response
`(class I compounds, see below) (12). Subsequent experiments were
`conducted with the compounds atthree concentrations: their ap-
`proximate TNF-a IC.9; 3 times the TNF-c IC5o; and 10 times the
`TNF-a IC59. Rolipram, a known TNF-a inhibitor (19), was used as
`a control. The effect of these drugs on other LPS-induced cyto-
`kines wasalso investigated. Compounds were addedat the men-
`tioned concentrations to LPS-stimulated human PBMC,andcyto-
`kine secretion into the culture supernatant was evaluated. Fig.
`1
`shows the effect of the drugs on the production of TNF-a , IL-1B,
`IL-6, IL-8, IL-10, and IL-12. Compounds clearly segregated into
`two different classes according to their effects on LPS-induced
`IL-1, IL-6, IL-10, and IL-12 cytokines. Oneclass of compounds
`(class I) showedsignificant inhibition of IL-1 B, at their TNF-a
`ICs9, and almost complete inhibition at higher concentrations,
`whereas compounds from class II had a more modest inhibitory
`effect, albeit significant at the higher concentrations (Fig. 1). Sim-
`ilarly, class I compounds significantly inhibited IL-6 levels,
`whereasclass II compoundsdid notaffect IL-6 production (Fig. 1).
`IL-8 levels were not significantly affected by either class of com-
`pounds, although class I showed a very minor trend toward inhi-
`bition of IL-8 production (Fig. 1).
`
`Table I. TNF-a and PDE4inhibition
`
`
`ICs9 Values (um)*
`
`
`
`
`
`8107‘81IsNsNYyUoysanBAq/es0"TounuTUNMamy//:dyywopepeojumog
`
`
`
` Compound TNF-a PDE4
`
`
`Thalidomide
`194
`>500
`CI-A
`0.01
`>100
`CI-B
`0.10
`>100
`CI-C
`0.04
`>100
`CIL-A
`12.6
`9.4
`CU-B
`20.6
`15.0
`CU-C
`0.21
`0.04
`
`Rolipram 0.40 0.15
`
`“TNF-a ICsq values were determined in LPS-stimulated human PBMC from
`dose-response curves derived from four independent experiments with different do-
`nors. PDE4 ICs values were determined in U937-purified enzyme from dose-response
`curves derived from three independent experiments. IC; values were calculated by
`nonlinear regression analysis (variable slope) using Prism by GraphPad Software.
`
`ALVOGEN, Exh. 1009, p. 0003
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`ALVOGEN, Exh. 1009, p. 0003
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`

`

`382
`
`THALIDOMIDE ANALOGUESAS DIFFERENTIAL CYTOKINE MODULATORS
`
`IL-4
`
`B
`
`r
`
`|
`
`—1— CILLA
`—~s— CIB
`—o— CILC
`—o—Cl-A
`—2— CIB
`ATOl
`~—x— Rolipram
`
`ua
`t
`
`400.
`
`75
`
`504
`
`254
`
`TNF-o
`100-
`
`+
`
`75
`
`504
`
`254
`
`od ot
`
`IL-6
`
`tr
`
`IL-8
`
`&.
`
`75-4 Qe 75-4
`50+
`;
`50+
`
`25 -
`
`
`
`
`
`LPS-inducedCytokineProduction(%activity)
`100+
`
`25 +
`
`IL-10
`
`400-
`
`300 +
`
`200+
`
`
`
`IL-12
`
`
`
`Concentration of Compounds
`(multiples of TNF-c. IC,,)
`
`FIGURE 1. Effect of thalidomide analogues on LPS-induced cytokines.
`PBMC wereculturedin triplicate and stimulated with LPS in the absence
`or presence ofthe indicated concentrations of thalidomide analogues. Su-
`pernatants were collected at 20 h after incubation and tested for cytokine
`levels by ELISA. Results, expressed as percentage of activity, are aver-
`ages = SEMofthree to five independent experiments with different do-
`nors. Drugs were tested at their TNF-a ICs, 3-fold TNF-a ICs, and 10-
`fold TNF-a ICs9. Class I compounds(closed symbols) inhibited IL-1 and
`IL-12 and stimulated IL-10 levels significantly (p < 0.001) at all concen-
`trations. IL-6 levels were significantly inhibited (p < 0.001) at concentra-
`tions above the TNF-a IC. valuesfor these drugs. Class II compounds and
`rolipram (open symbols) inhibited IL-1 and IL-12 significantly only at
`3-fold their TNF-a ICs, or higher (p < 0.05). These compounds did not
`inhibit IL-6 or IL-8. CII-A, CII-B, and rolipram significantly (p < 0.05)
`increased IL-10 levels at the two higher concentrations.
`
`The effect of these compounds onthe levels of the antiinflam-
`matory cytokine IL-10 wasalso tested. All compounds, except for
`CII-C, significantly increased IL-10 production. However, IL-10
`stimulation by class | compounds was clearly more extensiveatall
`concentrations (Fig. 1).
`
`—@— Thalidomide
`3501 —®-CIA
`—e—CI-B
`3004 —O—CIIA
`—A— II-B
`
`250
`
`200
`
`150
`
`100
`
`
`
`
`
`
`
`
`
`
`
` onQ ('H]-ThymidineIncorporation(cpmx10°) 0
`
`0
`
`100
`
`10
`1
`0.1
`0.01
`Concentration of Compounds (uM)
`FIGURE 2. Effect of thalidomide and analogueson theproliferative re-
`sponses of T cells. Purified (>97% CD3*)T cells were cultured in trip-
`licate and stimulated by 10 yg/ml immobilized anti-CD3 in the absence or
`presence of the indicated concentrations of thalidomide and analogues.
`[SH]Thymidine incorporation was measured for the last 18 h of 120-h
`cultures. Results, expressed as cpm, are averages + SEM offive indepen-
`dent experiments with different donors. Thalidomide and class I com-
`pounds increased cell proliferation significantly (p < 0.005). Cell prolif-
`eration wassignificantly inhibited by CU-B (p < 0.05).
`
`
`
`
`
`8107‘8IsnsnyUoyson3Aq/810"jounumTMMa//:cqyWoypepeoyumoq
`
`LPS-induced IL-12 levels were significantly inhibited by both
`classes of compoundsat the higher concentrations, but class I com-
`pounds were more potent (Fig. 1). Thus, in summary, compounds
`from class I caused a more pronounced inhibition of LPS-induced
`IL-1 and IL-12 in addition to the inhibition of IL-6 and a much
`greater stimulation of IL-10. Class If compounds showed signifi-
`cant inhibitory activities against LPS-induced IL-1 B and IL-12 but
`only at concentrations above their TNF-a ICs values. A modest
`but consistent stimulation of IL-10 was observed for class II com-
`pounds CII-A and CU-B. Rolipram, used as a control, showed
`cytokine-modulatory profiles comparable with those ofclass II
`compounds(Fig. 1).
`
`Effect of class I and class IT compounds on T cell-proliferative
`responses to immobilized anti-CD3 mAb
`OptimalT cell activation requires two types of signals (20). Signal
`1 is delivered byclustering ofthe T cell Ag receptor-CD3 complex
`through engagementof specific foreign peptides bound to MHC
`molecules on the surface of an APC. Signal 1 can be mimicked by
`cross-linking the TCR complexes with anti-CD3 mAb. Signal2 (or
`costimulation) is Ag independent and may be provided by cyto-
`kines or by surface ligands on the APC that interact with their
`receptors on the T cell. Costimulatory signals are essential to in-
`duce maximal T cell proliferation and secretion of cytokines in-
`cluding IL-2 which ultimately drives T cell clonal expansion (20).
`Thalidomide was recently reported to provide a costimulatory
`signal to T cells receiving primary stimulation via the TCR, re-
`sulting in increased cytokine production and proliferation (13). We
`now examined the effect of the two classes of thalidomide ana-
`logues on the proliferative responses ofpurified T cells stimulated
`by anti-CD3 mAb. Two compoundsfrom each class were tested in
`these assays. Again, the two classes of compounds showed differ-
`ential activities. Compounds from class II exerted a modest inhi-
`bition of T cell proliferation in response to immobilized anti-CD3,
`significant for only one of the two compounds (Fig. 2). Class I
`compounds, however, were potent costimulators of T cells and
`increasedcell proliferation significantly in a dose-dependent man-
`ner. As expected, thalidomide wasalso significantly costimulatory
`in this assay but not as potent. There was no proliferative response
`
`ALVOGEN, Exh. 1009, p. 0004
`
`ALVOGEN, Exh. 1009, p. 0004
`
`

`

`383
`
`Effect ofclass I and class II compounds on PDEA activity
`PDE4is one of the major phosphodiesterase isoenzymes found in
`human myeloid and lymphoid lineage cells. The enzyme plays a
`crucial role in regulating cellular activity by degrading the ubiq-
`uitous second messenger cAMP and maintaining it at low intra-
`cellular levels (21). Inhibition of PDE4 and the consequent in-
`creased cAMPlevels result in the modulation of LPS-induced
`cytokines including inhibition of TNF-a. As previously reported,
`class Il compounds, similarly to rolipram, are potent PDE4 inhib-
`itors (14). Therefore, we examined the effect ofclass I compounds
`on PDE4activity in purified fractions of the monocytic cell line
`U937. These compounds did not show significant PDE4 inhibitory
`activity at up to 100 4M (Table I). These results strongly suggest
`that the molecular target of the class I compoundsis not PDE4.
`Thus, class I compoundsconstitute a new class of immunomodu-
`lators. These compoundsare efficient TNF-« inhibitors but do not
`act as PDE4 inhibitors. Unlike PDE4 inhibitors, which usually
`decrease T cell activity, class I compoundsare potent stimulators
`of T cell proliferation and IFN-y and IL-2 production.
`
`The Journal of Immunology
`
`—®— Thalidomide
`—@—CIA
`—E— CI-B
`—O— CIA
`
`IFNy
`
`—4S— CIL-B CytokineLevels(pg/ml)
`
`
`
`
`
` 0
`
`100
`70
`i
`O4
`601
`0
`Concentration of Compounds (uM)
`FIGURE 3. Effects of thalidomide and analogues on T cell cytokine
`production. Purified T cells (for IFN-y determination) or PBMC(for IL-2
`determination) were cultured in triplicate and stimulated with immobilized
`anti-CD3 in the absence or presence of the indicated concentrations of
`thalidomide and analogues. IFN-y levels and IL-2 levels were determined
`by ELISA. Results, expressed as picogramsper milliliter of cytokine, are
`averages + SEM ofthree independent experiments with different donors.
`Thalidomide and class I compoundsincreased IL-2 and IFN-y production
`significantly (p < 0.05 and p < 0.01, respectively).
`
`
`
`810Z“8TIsnBnVyuoysondfq/3x0"jounwUnlaMay:dyyWOpopeojumoq
`
`Differential effects ofclass 1 compounds onTcell-dependent and
`T cell-independentcytokine production
`IL-12 is produced primarily by APC (monocytes/macrophages and
`dendritic cells) and is regulated by both T cell-dependent and T
`cell-independent pathways. LPS induction of IL-12 is an example
`of the T cell-independent pathway. In the T cell-dependent path-
`way, on the other hand, the production of IL-12 is induced pri-
`marily by the interaction of CD40L on activated T cells with CD40
`on IL-12-producing APC (22, 23). To study the effect of thalido-
`mide and class I compounds on cytokine production in a T cell-
`dependent system, PBMC werestimulated through the TCR with
`immobilized anti-CD3 mAb, and IL-12, TNF-a, and IL-10 were
`measured. In this system, both thalidomide and the class I com-
`pound CI-A induced significant increases in IL-12 production (Fig.
`4). However, thalidomide did not affect the production of TNF-a
`and IL-10 by anti-CD3. On the other hand, the class I drug CI-A
`slightly stimulated TNF-a production but significantly inhibited
`IL-10 production in this system.
`We next examinedthe effect of thalidomide and two class I
`compoundson the expression of CD40L on T cells stimulated by
`anti-CD3. Thalidomide and class I compounds induced a dose-
`dependentand significant increase in CD40L expression that par-
`alleled the increases in IL-12 production induced by anti-CD3
`(Fig. 5).
`Wealso tested the effect of the drugs on T-cell independent
`IL-12 production. PBMC werestimulated with LPS in the pres-
`ence and absenceofthe drugs, and IL-12, TNF-a, and IL-10 levels
`were determined. LPS-induced IL-12 and TNF-a levels were sig-
`nificantly inhibited by thalidomide and by the class I drug CI-A,
`whereas IL-10 wassignificantly stimulated (Fig. 4). Similar results
`were obtained with another class I compound, CI-B (data not
`shown). Thus, class I compounds modulate IL-12, TNF-a, and
`IL-10 production differently in cells from the same donor depend-
`ing on whether the stimulus is directed at the monocytes/macro-
`phages (LPS)or T cells (anti-CD3).
`
`to these drugs in the absence of anti-CD3, indicating that these
`drugs are not mitogenic per se but provide a secondary, costimu-
`latory signal (data not shown).
`Thus, whereas class I compounds had a thalidomide-like co-
`stimulatory effect on the proliferative responses of T cells, com-
`pounds from class II were modestinhibitors. Rolipram, used as a
`control, modestly
`inhibited
`the
`proliferation
`of
` anti-
`CD3-stimulated T cells, similarly to class IT compounds(data not
`shown).
`
`Effect ofclass I and class I compounds on T cell cytokine
`production
`Wenext evaluated the effect of the two classes of compounds on
`production of the T cell cytokines IL-2 and IFN-y. Class I com-
`pounds induced significant concentration-dependent increases in
`IFN-y at 72 h [peak of the production ofthis cytokine in this
`system (13)] (Fig. 3). Class Il compounds, onthe other hand,either
`had noeffect orslightly inhibited IFN-- production at higher drug
`concentrations. As reported previously, thalidomide significantly
`stimulated IFN-y production, althoughit required higher dosages
`(2-3 logs of magnitude) for activity.
`Similarly, IL-2 production was significantly increased in anti-
`CD3-stimulated PBMCby class I compounds, whereas class II
`compounds showednoeffect (Fig. 3). Thalidomide stimulated IL-2
`production significantly but only at higher doses.
`Thus, class I compounds were found to be efficient T cell co-
`stimulators leading to the augmented production of the T cell cy-
`tokines IL-2 and IFN-y.
`
`
`
`Discussion
`
`To develop analogues of thalidomide with increased anti-TNF-o
`potency and reduced or absentteratogenic potential, a program to
`identify improved TNF-a inhibitors was initiated. Here we report
`that when a selected group ofthese TNF-a-inhibitory compounds
`wasfurther characterized, a dichotomous pattern in cytokine mod-
`ulation activities was revealed. Although all tested compounds
`ALVOGEN, Exh. 1009, p. 0005
`
`ALVOGEN, Exh. 1009, p. 0005
`
`

`

`384
`
`THALIDOMIDE ANALOGUESAS DIFFERENTIAL CYTOKINE MODULATORS
`
`|
`
`| —e—Cl-A
`
`—m—Cl-B
`asa, We
`
` sher
`
`0.01
`
`O41
`
`0.001
`0
`100
`10
`Concentration of Compounds (uM)
`FIGURE 5. Effect ofthalidomide andtwoclass I compounds on CD40L
`expression by CD3* cells in PBMC stimulated by anti-CD3. Cells were
`treated with thalidomide and analogues and harvested for two-color cyto-
`metric analysis at 48 h. Results, expressed as percentage ofcells staining
`for CD40L, are averages + SD from four independent experiments with
`different donors. All three drugs increased CD40L expression significantly
`(p < 0.05).
`
`—
`
`BO ce Thalidomide
`
`NmOo
`
`wears
`
`(%) o
`
`CD40LExpression
`
`0
`
`1
`
`
`
`—4— anti-CD3
`—4—LPS
`
`1
`
`|
`
`
`
`150
`
`200
`
`IL-12s&
`
`
`
`CytokineProduction(%activity)
`
`2004
`
`A 150
`i} 7
`
`100
`
` |
`|
`F
`
`
`
`
`
`to 70 1 140 100 ° 0 001 .01 1
`
`
`
`
`Thalidomide
`CI-A
`Concentration (uM)
`FIGURE 4. Effect of thalidomide andclass I compound CI-A on anti-
`CD3 and LPS-induced TNF-a, [L-12, and IL-10 levels. PBMCwerestim-
`ulated with anti-CD3 or LPS andtreated with thalidomide and Class I-A at
`the indicated concentrations. LPS-induced cytokines were determined at
`24 h after stimulation. Anti-CD3-induced TNF-a was determined at 24 h;
`anti-CD3 induced IL-12 and IL-10 levels were determined at 72 h after
`stimulation. Results, expressed as percentageof activity, are averages +
`SEMofthree to six independent experiments with different donors. Tha-
`lidomide and CI-A increased anti-CD3-induced IL-12 production signifi-
`cantly (p < 0.05), whereas LPS-induced IL-12 and TNF-a levels were
`significantly inhibited by the drugs (p < 0.01 and p < 0.05, respectively).
`LPS-induced IL-10 levels were increased by thalidomide and CI-A (p <
`0.05) whereas anti-CD3-induced IL-10 was significantly inhibited by CI-A
`only (p < 0.05). No IL-12, IL-10, or TNF-a was detected in cultures with
`or without added compoundsin the absence of LPS or anti-CD3 (data not
`shown).
`
`were much more potent TNF-a inhibitors than the parent drug
`thalidomide,
`they differed in the slope of their dose-response
`curves as well as in the modulation of other monocyte and lym-
`phocyte cytokines. Membersofone class of compounds, referred
`to here as class I, were broad inhibitors of the LPS-induced proin-
`flammatory monocyte cytokines TNF-a , IL-1, IL-6, and IL-12
`while potently augmenting the secretion of the antiinflammatory
`cytokine IL-10. Class Il compounds, onthe other hand, inhibited
`
`S107“81IsNSNYUOsandAq/810"ounMMA//:dyyWOpoproyuMoc
`
`
`
`both TNF-a and IL-12 in LPS-stimulated PBMC but had little
`effect on the production of other LPS-induced monocyte cytokines
`such as IL-1, IL-6, or IL-8. The latter drugs also produced a
`modest stimulation of LPS-induced IL-10 levels. In all of these
`effects, class If compoundsclosely resemble thalidomide (12).
`Recently, we reported that thalidomide provides a costimulatory
`signal to T cells, resulting in increased T cell proliferation and
`augmented IL-2 and IFN-y production (13). Resting T cells require
`a costimulus in addition to the primary signal mediated by the T
`cell Ag receptor to achieve optimal activation (20). Such a co-
`stimulus alone will not activate the T cell. Similarly to thalido-
`mide, class I compoundsalso exhibited T cell costimulatory prop-
`erties but were far more potent than the parent molecule in this
`respect. Thus, these compounds caused marked increases in pro-
`liferation and secretion of IL-2 and IFN-y by anti-CD3-stimulated
`T cells. In the absence ofthe TCR-mediated stimulus, however,the
`drugs had noactivating effect. Costimulation by class I compounds
`also resulted in increased CD40L expression onT cells, associated
`with enhanced T cell-dependent IL-12 production. These findings
`show that in addition to their strong antiinflammatory properties,
`class | compoundsefficiently costimulate T cells, achieving both
`effects with 100 to 1000 times the potency of thalidomide.
`The different cytokine-modulatory profiles of the two classes of
`compoundsare likely to be related to their molecular targets. Class
`Tl compoundsare potent inhibitors of PDE4 (14). PDE4inhibition
`leads to increases in intracellular cAMP levels resulting in the
`suppression of TNF-a and IL-12 production and increased pro-
`duction of the antiinflammatory cytokine IL-10 (24, 25). IL-6 and
`IL-8, on the other hand, are not directly regulated by cAMP(25,
`26). IL-1 is only partially affected by inhibition ofPDE4 (26,27).
`Thus, PDE4 inhibitors appear to have a selective antiinflammatory
`action. It is also well established that raising cAMPlevels in T
`cells during the early phase of mitogen or Agactivation results in
`a decrease in proliferative potential (28, 29). Indeed, class II com-
`pounds modestly but consistently inhibited T cell proliferation and
`T cell cytokine production,
`in accord with rolipram and other
`known PDE4inhibitors (29, 30). In addition, class II compounds
`either inhibited or had no effect on CD40L expression on T cells.
`Although there are no reports on the effects of PDE4 inhibitors on
`CD40L expression, other cAMP-elevating agents have been shown
`to be unable to induce CD40L expression on T lymphocytes (31).
`ALVOGEN, Exh. 1009, p. 0006
`
`ALVOGEN, Exh. 1009, p. 0006
`
`

`

`
`
`
`
`8107‘SIsndnyUoysondAq/810'jounuTTMMM/:dyyyWOTpepeojuMog
`
`
`
`
`
`385
`
`Acknowledgments
`Wethank Judy Adamsfor help in preparing the figures, Marguerite Nulty
`for help with preparing the manuscript, and Dr. Victoria H. Freedman for
`critical discussions during the preparation of this report.
`
`References
`
`N
`
`we
`
`1. Kindler, V., A. P. Sappino, G. E. Grau,P. F. Piguet, and P. Vassalli. 1989. The
`inducing role of tumor necrosis factor in the developmentofbactericidal gran-
`ulomas during BCGinfection. Cell 56:73.
`J. Triebold, K. Pfeffer,
`. Flynn,
`J.