. BUHLHMXHHBoBIEs‘H'réf}Hiv}9BwR
`
` fig-PD—
`
`: CT 2.
`
`
`EmsEKH’c H
`
`
`Differential Cytokine Modulation and T Cell
`
`firejournal qr
`Immunology Activation by Two Distinct Classes of
`Thalidomide Analogues That Are Potent
`Inhibitors of TNF- 06
`
`This information is current as
`of August 18, 2018.
`
`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
`
`Jlmmunol 1999; 163:380-386; ;
`http://www.jimmunol.org/content/l 63/1/3 80
`
`810K‘81isnfinvuolsQnB({q/8.10'10unwuitf'MMM//zduqwonpopcolumoq
`
`—\
`
`References
`
`This article cites 44 articles, 11 of which you can access for free at:
`http://www.jimmunol.org/content/ 1 63/ 1/3 80.full#ref—list-1
`
`Why The JI? Submit online.
`
`~ Rapid Reviews! 30 days* from submission to initial decision
`
`- No Triage! Every submission review
`
`- Fast Publication! 4 weeks from acce ed by practicing scientists
`
`ptance to publication
`
`*average
`
`Subscription
`
`Information about subscribing to The Journal ofImmunology is online at:
`http://j immunol.org/subscription
`
`Permissions
`
`Submit copyright permission requests at:
`http://wwwaai.org/About/Publica
`tions/JI/copyright.html
`
`Email Alerts Receive free email-alerts when ne
`http://jimmunol.org/alerts
`
`w articles cite this article. Sign up at:
`
`-\
`The Journal ofImmunology 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
`
`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-a1
`
`Laura G. Corral,“ Patrick A. J. Haslettf George W. Muller,* Roger Chen,* Lu-Min Wong,*
`Christopher J. Ocampof Rebecca T. Patterson,* David I. Stirling,* and Gilla Kaplan“f
`TNF-a mediates both protective and detrimental manifestations of the host immune response. Our previous work has shown
`thalidomide to be a relatively selective inhibitor of TNF—a production in vivo and in vitro. Additionally, we have recently reported
`that thalidomide exerts a costimulatory etfect on T cell responses. To develop thalidomide analogues with increased anti-TNF-ae
`activity and reduced or absent toxicities, novel TNF—a inhibitors were designed and synthesized. When a selected group of these
`compounds was examined for their immunomodulatory activities, difl'erent patterns of cytokine modulation were revealed. The
`tested compounds segregated into two distinct classes: one class of compounds, shown to be potent phosphodiesterase 4 inhibitors,
`inhibited TNF—oz production, increased IL-10 production by LPS-induced PBMC, and had little 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-lB, and IL—6 and greatly increased IL—10
`production by LPS-induced PBMC. Similar to thalidomide, the effect of these agents on IL—12 production was dichotomous; IL-12
`was inhibited when PBMC were stimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The
`latter elfect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatory activities of these classes
`of thalidomide analogues may potentially allow them to be used in the clinic for the treatment of different immunopathological
`disorders. The Journal of Immunology, 1999, 163: 380—386.
`
`umor necrosis 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—or is essential for
`granuloma formation and the control of bacterial 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
`RNA viruses (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 bowel disease after treatment of patients
`with neutralizing anti~TNF-a Abs (5, 6). Additionally, elevated
`levels of TNF-oz have been associated with the fevers, malaise, and
`weight loss that accompany chronic infections (7), and reductions
`in TNF-oz levels have been linked with an amelioration of clinical
`symptoms in a number of disease states (8—11).
`Our previous work has shown thalidomide to be a relatively
`selective inhibitor of TNF—or 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-or levels with a concomitant abrogation of clinical symptoms
`
`
`*Cclgene 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 of this 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.
`
`I This work was supported by Cclgenc Corporation. GK was supported by Public
`Health Service National Institutes of Health Research Grant AI~22616; PAJH. was
`supported by General Clinical Research Center Grant MOl-RR00102.
`2 Address correspondence and reprint requests to L. G. Corral, Laboratory of Cellular
`Physiology and Immunology, Rockefeller University, 1230 York Avenue, New York,
`NY 10021. E—mail address; corrall@roekvax.rockefelleredu
`
`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 mRNA levels in association with an accelerated weight
`gain (8). In vitro, thalidomide has been shown to selectively par-
`tially (50—70%) inhibit TNF-oz produced by monocytes and mac—
`rophages stimulated 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 eificient TNF-or inhibitors than
`thalidomide, structural analogues of the parent molecule have been
`synthesized and examined for inhibition of TNF—oz production. Re-
`cently, some of these thalidomide analogues have been described
`(l2, 14, 15, 47). On a molar basis, these reported compounds are
`up to 50,000-fold more potent
`than thalidomide at
`inhibiting
`TNF-a production by PBMC in vitro. 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
`
`PBMC were isolated from the blood of healthy volunteers by Ficoll—
`Hypaque (Pharmacia, Piscataway, NJ) density ccntrifugation as previously
`described (12). T lymphocytes were purified from PBMC by rosetting with
`neuraminidase—treated sheep erythrocytes and subsequent incubation of
`erythrocyte-resetting cells on a nylon wool column. Nonadherent cells
`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
`
`ALVOGEN, EXh. 1009, p. 0002
`
`
`
`3103‘SIisanVuoisonfiAq/310'[0UHUIIIII[‘MAAAA/fiduqurea}popeommoq
`
`
`
`
`
`ALVOGEN, Exh. 1009, p. 0002
`
`

`

`The Journal of Immunology
`
`381
`
`10% AB l human serum, 2 rnM L-glutamine, 100 U/ml penicillin, and 100
`ug/ml streptomycin (Life Technologies).
`
`Thalidomide and analogues
`
`Thalidomide and analogues (Cclgenc, 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 following structural analogues were used: CII—A is
`compound 3a (14) and CC-1069 (12); Cll-B is compound CC-3052 (16)
`with the carboxymethyl group replaced by an amide moiety; CIl-C is an
`amino—substituted analogue of compound 4b (14); compounds CI-A, CI—B,
`and CI—C are amino-substituted analogues of thalidomide. CI-A is 5a, CI—B
`is 8a and CI-C is 14 (47).
`
`PBMC stimulation by LPS
`
`PBMC (2 X 105 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—a , IL-1 3, IL—6, IL—8, IL-10, and lL-l2 (12).
`Cells were incubated with or without thalidomide or analogues for 20 h,
`and supematants were collected for the determination of cytokine levels by
`ELISA.
`
`PBMC stimulation by anti—CD3 Ab
`
`PBMC (l X 106 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, NI) as previously de-
`scribed (13). The anti-CD3 Ab was diluted to 10 pig/ml in 100 Ml 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 dilutions of thalidomide and analogues were added at the start
`of the cell cultures. Supernatants were collected at 24, 48, and 72 h and
`assayed for IL—10, IL-l2, and "INF—or levels. Cells were collected at 48 h
`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 and proliferation assays
`
`Purified T cells (2 X 105 cells/well) in 96-well Costar tissue 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 [3H]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 [3H]thy—
`midine incorporation was measured with a LKB 1205 Betaplate liquid
`scintillation counter (Wallac, Gaithersburg, MD)
`
`Phosphodiesterase 4 (PDE4) inhibition assay
`
`
`
`PDE4 inhibition was evaluated in purified extracts of promonocytic U937
`cells using a modified method of Hill and Mitchell (17) as previously
`described (14). Cells (l X 109) were washed in PBS and lysed in cold
`homogenization bu'Z’er (20 mM Tris—HCl, pH 7.1; 3 mM 2—ME;
`1 mM
`MgCl; 0.1 mM EGTA,
`1 MM PMSF, 1 lug/ml leupeptin). After homoge—
`nization with a Douncc homogenizer, the supernatant was collected by
`centrifugation and loaded onto a Sephacryl 8—200 column equilibrated with
`homogenization bu’fer. PDE4 was eluted in homogenization buffer, and
`enzyme activity was determined in 50 mM Tris-HCl, pH 7.5, 5 mM MgC12,
`and 1 MM cAMP (of which 1% was [3H]cAMP) as described in detail by
`Thompson et a1. (18). Reactions were performed at 30°C for 30 min and
`terminated by boiling for 2 min. Briefly. cyclic 3’,5’-[3H]AMP was con-
`verted to 5’-[3H]AMP by phosphodiesterase. The separation of 5'-
`[3H]AMP from 3’,5’-[3H]AMP was achieved by enzymatically converting
`5’—[3H]AMP to [3H]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 ,ul ofAGl-X8 resin
`(Bio-Rad, Hercules, CA) that absorbs cyclic 3’,5’-[3H]AMP. Samples were
`then spun at 3000 rpm for 5 min, and 50 ptl 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.
`IC50 values were determined from dose-re-
`sponses curves derived from at least three independent experiments done in
`
`
`3 Abbreviations used in this paper: CD40L, CD40 ligand; PDE4, phosphodiesterase 4.
`
`duplicate. IC50 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—oz , IL—lfi,
`IL—2, lL—6,
`lL—S, lL-10, IL—12 (p40 and p70), and lFN—y levels were mea-
`sured by ELISA (Endogen, Cambridge, MA) as described by the
`manufacturer.
`
`Statistical analysis
`
`Data were evaluated by the Friedman test, a nonparametric ANOVA, in
`View of the small sample size. The SPSS computer program was used.
`Significance was set at p < 0.05.
`
`Results
`
`Eject of thalidomide analogues on LPS-induced cytokine
`production
`
`A group of thalidomide analogues were selected for their capacity
`to inhibit TNF—a production by LPS-stimulated PBMC. Their IC50
`values for TNF~or (the concentration at which each compound was
`able to inhibit TNF—a levels by 50%) were established when
`screening these agents (Table I). Although all 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 11 compounds, see below) (12). Other com—
`pounds, however, showed a flatter, thalidomide—like dose response
`(class I compounds, see below) (12). Subsequent experiments were
`conducted with the compounds at three concentrations: their ap-
`proximate TNF—a ICSO; 3 times the TNF-a ICSO; and 10 times the
`TNF-a ICSO. Rolipram, a known TNF-a inhibitor (19), was used as
`a control. The eifect of these drugs on other LPS—induced cyto-
`kines was also investigated. Compounds were added at the men-
`tioned concentrations to LPS—stimulated human PBMC, and cyto—
`kine secretion into the culture supernatant was evaluated. Fig.
`1
`shows the elfect of the drugs on the production of TNF—a , lL-lB,
`IL-6, lL-8, IL-10, and IL—12. Compounds clearly segregated into
`two different classes according to their effects on LPS-induced
`IL—1 3, IL-6, lL-lO, and IL—12 eytokines. One class of compounds
`(class 1) showed significant inhibition of IL—18, at their TNF-a
`ICSO, 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,
`whereas class II compounds did not afieet IL-6 production (Fig. 1).
`lL-8 levels were not significantly affected by either class of com-
`pounds, although class 1 showed a very minor trend toward inhi-
`bition of IL-8 production (Fig. 1).
`
`Table I. TNF-oz and PDE4 inhibition
`
`
` Compound ”INF-or PDE4
`
`IC50 Values Um)“x
`
`Thalidomide
`194
`>500
`CI—A
`0.01
`> 100
`Cl—B
`0. 1 0
`> 1 00
`CI—C
`0.04
`>100
`CH-A
`12 .6
`9.4
`Cll—B
`20.6
`15.0
`ClI-C
`0.21
`0.04
`
`Rolipram 0.40 0.15
`
`
`
`
`‘1 TNF-a ICSD values were determined in LPS—stimulated human PBMC from
`dose—response curves derived from four independent experiments with different do-
`nors. PDE4 IC50 values were determined in U937—purified enzyme Erom dose—response
`curves derived from three independent experiments. IC50 values were calculated by
`nonlinear regression analysis (variable slope) using Prism by GraphPad Software.
`
`ALVOGEN, Exh. 1009, p. 0003
`
`
`
`
`
`3103‘81isnfinvuo1s9n8[{qy/Sio'iountuuirl'AAMAA//zdnqwortpaprzommoq
`
`ALVOGEN, Exh. 1009, p. 0003
`
`

`

`382
`
`THALIDOMIDE ANALOGUES AS DIFFERENTIAL CYTOKINE MODULATORS
`
`250
`
`200
`
`150
`
`100
`
`
`
` (IIC [3H]‘ThymidineIncorporation(cpmx106)
`
`‘O— Thalidomide
`350 + Cl-A
`—I— 01-8
`~D— Cll-A
`-—A- CIl-B
`
`300
`
`
`
`
`
`
`
`
`
`O
`0
`0.01
`0.1
`1
`10
`100
`Concentration of Compounds (pM)
`
`
`
`
`
`3[OZ‘gr1sn3nvuotsonfiAq/3J0‘rountuturf'MMM//zchmLuci}popeorumoq
`
`FIGURE 2. Eifect of thalidomide and analogues on the proliferative re—
`sponses of T cells. Purified (>97% CD3 +) T cells were cultured in trip—
`licate and stimulated by 10 ug/ml immobilized anti-CD3 in the absence or
`presence of the indicated concentrations of thalidomide and analogues.
`[3H]Thymidine incorporation was measured for the last 18 h of l20-h
`cultures. Results, expressed as cpm, are averages : SEM of five indepen-
`dent experiments with diiferent donors. Thalidomide and class I com—
`pounds increased cell proliferation significantly (p < 0.005). Cell prolif-
`eration was significantly inhibited by CII—B (p < 0.05).
`
`LIPS—induced IL—12 levels were significantly inhibited by both
`classes of compounds at 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-lB and IL-12 in addition to the inhibition of IL-6 and a much
`greater stimulation of IL~10. Class II compounds showed signifi-
`cant inhibitory activities against LPS-induced IL—1 [3 and IL—12 but
`only at concentrations above their TNF—a IC50 values. A modest
`but consistent stimulation of IL—10 was observed for class II com—
`pounds ClI-A and CII-B. Rolipram, used as a control, showed
`cytokine-modulatory profiles comparable with those of class II
`compounds (Fig. 1).
`
`Efect of class I and class II compounds on T cell—proliferative
`responses to immobilized anti—CD3 mAb
`
`Optimal T cell activation requires two types of signals (20). Signal
`1 is delivered by clustering of the T cell Ag receptor-CD3 complex
`through engagement of 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. Signal 2 (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 of purified T cells stimulated
`by anti-CD3 mAb. Two compounds from 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
`increased cell proliferation significantly in a dose—dependent man—
`ner. As expected, thalidomide was also significantly costimulatcry
`in this assay but not as potent. There was no proliferative response
`
`ALVOGEN, EXh. 1009, p. 0004
`
`—D—Cll-A
`——A——Cll—B
`00_TNF-or “We”
`-—-O—Cl-A
`
`won-113
`
`754
`
`50-
`
`254
`
`DJ
`
`—-—cr-B
`”MC"?
`——><—-Rohpram
`
`r
`
`T
`
`l
`
`75-
`
`504
`
`25-
`
`T
`
`t
`
`T
`
`
`
`o+—r———-—1——.—r~—
`
`lL-8
`
`100— 1%
`
`504
`
`_
`
`25
`
`O~~—r——,—.—r_
`
`IL-12
`
`
`
`2:.
`
`g
`
`iL—B
`
`S 3
`
`%
`
`Om
`
`75—
`
`50—
`
`-
`
`IL-tO
`
`3% 100‘ ‘fifi
`.9
`t:
`\E
`3
`'D
`*5
`\E 75—
`9
`C
`3‘];
`9%
`S
`O 25
`‘DG)
`8
`'O O’hf—r-“fi—«e
`,2
`c}:
`E
`
`400—
`
`300—
`
`2001
`
`1004
`
`
`
`
`
`100
`
`75
`
`so
`
`
`
`25}
`
`o
`
`3
`
`1 0
`
`
`i
`
`Concentration of Compounds
`(multiples of TNF~a i050)
`
`FIGURE 1. Effect of thalidomide analogues on LPS—induced cytokines.
`PBMC were cultured in triplicate and stimulated with LPS in the absence
`or presence of the 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 : SEM of three to five independent experiments with different do—
`nors. Drugs were tested at their TNF—a ICSO, 3—fold TNF-a ICSO, and 10-
`fold TNF~01 ICSO. Class I compounds (closed symbols) inhibited IL-1 [3 and
`IL—lZ and stimulated IL-lO levels significantly (p < 0.001) at all concen—
`trations. IL—6 levels were significantly inhibited (p < 0.001) at concentra-
`tions above the TNF—rx IC50 values for these drugs. Class II compounds and
`rolipram (open symbols) inhibited IL—IB and IL-12 significantly only at
`3-fold their TNF-oz IC50 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—lO levels at the two higher concentrations.
`
`The eifect of these compounds on the levels of the antiinflam—
`matory cytokine IL-10 was also tested. All compounds, except for
`CII-C, significantly increased IL-10 production. However, IL-lO
`stimulation by class 1 compounds was clearly more extensive at all
`concentrations (Fig. l).
`
`ALVOGEN, Exh. 1009, p. 0004
`
`

`

`The Journal of Immunology
`
`383
`
`—0— Thalidomide
`—O— Cl-A
`——I— CI-B
`—-Cl— ClI—A
`
`800
`
`600
`
`400
`
`200 CytokineLevels(pg/ml)
`
`
`
`
`
`
`
` o
`
`0.01
`
`0.1
`
`1
`
`to
`
`100
`
`Concentration of Compounds (pM)
`
`FIGURE 3. Eifects of thalidomide and analogues on T cell cytokine
`production. Purified T cells (for IFN-y determination) or PBMC (for IL-2
`determination) were cultLu‘ed 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 lL—2 levels were determined
`by ELISA. Results, expressed as picograms per milliliter of cytokine, are
`averages i SBM of three independent experiments with diiferent donors.
`Thalidomide and class I compounds increased IL-2 and IFN-y production
`significantly (p < 0.05 and p < 0.01, respectively).
`
`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 elfect on the proliferative responses of T cells, com-
`pounds from class II were modest inhibitors. Rolipram, used as a
`control, modestly
`inhibited
`the
`proliferation
`of
`anti-
`CD3~stimulated T cells, similarly to class II compounds (data not
`shown).
`
`Eflect ofclass I and class 11 compounds on T cell cytokine
`production
`
`We next 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 11 [peak of the production of this cytokine in this
`system (13)] (Fig. 3). Class 11 compounds, on the other hand, either
`had no effect or slightly inhibited IFN—y production at higher drug
`concentrations. As reported previously, thalidomide significantly
`stimulated IFN-y production, although it required higher dosages
`(2—3 logs of magnitude) for activity.
`Similarly, IL-2 production was significantly increased in anti—
`CD3-stimulated PBMC by class I compounds, whereas class II
`compounds showed no etfect (Fig. 3). Thalidomide stimulated IL-2
`production significantly but only at higher doses.
`Thus, class I compounds were found to be eflicient T cell co-
`stimulators leading to the augmented production of the T cell cy-
`tokines IL—2 and lFN—y.
`
`
`
`Eflcct of class I and class [1 compounds on PDE4 activity
`PDE4 is 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 (2l). Inhibition of PDE4 and the consequent in-
`creased CAMP levels result in the modulation of LPS-induced
`cytokines including inhibition of TNF-oz. As previously reported,
`class II compounds, similarly to rolipram, are potent PDE4 inhib-
`itors (14). Therefore, we examined the efiect of class I compounds
`on PDE4 activity in purified fractions of the monocytic cell line
`U937. These compounds did not show significant PDE4 inhibitory
`activity at up to 100 MM (Table I). These results strongly suggest
`that the molecular target of the class I compounds is not PDE4.
`Thus, class 1 compounds constitute a new class of irnmunomodu—
`lators. These compounds are efficient TNF-a inhibitors but do not
`act as PDE4 inhibitors. Unlike PDE4 inhibitors, which usually
`decrease T cell activity, class I compounds are potent stimulators
`of T cell proliferation and IFN—y and IL-2 production.
`
`Di/j’erential ejects of class 1 compounds on T cell—dependent and
`T cell—independent Gyro/cine production
`
`IL—l2 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-l2—producing AFC (22, 23). To study the eifect of thalido—
`mide and class I compounds on cytokine production in a T cell-
`dependent system, PBMC were stimulated through the TCR with
`immobilized anti~CD3 mAb, and IL-12, TNF-a, and lL—lO were
`measured. In this system, both thalidomide and the class 1 com-
`pound CI-A induced significant increases in IL—12 production (Fig.
`4). However, thalidomide did not afiect the production of TNF-oz
`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—lO production in this system.
`We next examined the effect of thalidomide and two class I
`compounds on the expression of CD40L on T cells stimulated by
`anti—CD3. Thalidomide and class I compounds induced a dose-
`dependent and significant increase in CD4OL expression that par-
`alleled the increases in IL—12 production induced by anti-CD3
`(Fig. 5).
`We also tested the effect of the drugs on T-cell independent
`IL-12 production. PBMC were stimulated with LPS in the pres-
`ence and absence of the drugs, and IL—l2, TNF—oz, and IL—10 levels
`were determined. LPS-induced IL-12 and TNF-oz levels were sig-
`nificantly inhibited by thalidomide and by the class I drug CI—A,
`whereas IL-10 was significantly 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—lO 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).
`
`Discussion
`
`To develop analogues of thalidomide with increased anti—TNF—oz
`potency and reduced or absent teratogenic potential, a program to
`identify improved TNF-oz inhibitors was initiated. Here we report
`that when a selected group of these TNF—a-inhibitory compounds
`was further characterized, a dichotomous pattern in cytokinc mod-
`ulation activities was revealed. Although all tested compounds
`ALVOGEN, Exh. 1009, p. 0005
`
`
`
`8102?‘81isnfinVuoisonfiKq/fi10'[ountuun_l'mm/n//2dimwonpapeommoq
`
`ALVOGEN, Exh. 1009, p. 0005
`
`

`

`384
`
`THALIDOMIDE ANALOGUES AS DIFFERENTIAL CYTOKINE MODULATORS
`
`—A— anti»CD3
`—A— LPS
`
`25‘ —o— Thalidomide
`
`" + Ci—A
`
`-—-l
`
`-—L
`
`—-~Cl-B
`
`I
`
`l
`I?
`
`i-—I
`
`is, / —
`_. Ou
`
`
`
`
`
`CD40LExpression("/o)
`
`NO
`
`A
`
`‘7“
`
`
`
`
`
`
`
`
`
`8[oz‘8lanfinvno1son8xiq/3JO'IOUHIUIIIII‘MMM//Zd11q{110.1}papaoiumoq
`
`Dir—WWW ‘WW
`0
`1
`10
`100
`o ’
`0.001
`0.01
`0.1
`Concentration of Compounds (pM)
`
`FIGURE 5. Effect of thalidomide and two class 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 of cells staining
`for CD40L, are averages 1‘ SD from four independent experiments with
`diiferent donors. All three drugs increased CD401. expression significantly
`(p < 0.05).
`
`
`
`both TNF-oc and IL—IZ in LPS-stimulated PBMC but had little
`efect on the production of other LPS-induced monocyte cytokines
`such as IL—IB, IL-6, or IL-8. The latter drugs also produced a
`modest stimulation of LPS-induced IL—10 levels. In all of these
`eLTects, class II compounds closely resemble thalidomide (12).
`Recently, we reported that thalidomide provides a costimulatory
`s'gnal 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 primaly 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 compounds also 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 no activating eifect. Costimulation by class I compounds
`also resulted in increased CD40L expression on T cells, associated
`with enhanced T cell-dependent IL—12 production. These findings
`show that in addition to their strong antiinflammatory properties,
`class I compounds eificiently costimulate T cells, achieving both
`efiects with 100 to 1000 times the potency of thalidomide.
`The different cytokine—modulatory profiles of the two classes of
`compounds are likely to be related to their molecular targets. Class
`II compounds are potent inhibitors of PDE4 (14). PDE4 inhibition
`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
`lL-8, on the other hand, are not directly regulated by CAMP (25,
`26). IL-IB 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 CAMP levels in T
`cells during the early phase of mitogen or Ag activation 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 PDE4 inhibitors (29, 30). I11 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
`
`200
`
`200
`
`/
`
`
`
`\Efig 100— \
`
`150
`150
`/{
`100i ilk/“RE 1001 j
`
`l
`
`
`
` CytokineProduction(°/oactivity)
`
`
`150}
`fl/i 1501 /i/
`
`X 150
`150 j 100
`iL~12 a
`\
`RAE 50
`
`U10
`
`l
`
`200i
`
`O .
`
`2 0
`
`£4
`
`0
`
`‘i
`
`10
`
`100
`
`Ci-A
`Thalidomide
`Concentration (uM)
`
`FIGURE 4. Effect of thalidomide and class I compound CI—A on anti—
`CD3 and LPS-induced TNF-oc. IL—12, and IL-10 levels. PBMC were stim—
`ulated with anti-CD3 or LPS and treated with thalidomide and Class I-A at
`the indicated concentrations. LPS-induced cytokines were determined at
`24 h after stimulation. Anti-CD3—induced TNF—oz was determined at 24 h;
`anti-CD3 induced IL—lZ and IL-10 levels were determined at 72 h after
`stimulation. Results, expressed as percentage of activity, are averages i
`SEM of three 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—oz levels were
`significantly inhibited by the drugs (p < 0.01 and p < 0.05, respectively).
`LPS-induced lL—IO levels were increased by thalidomide and CI—A (p <
`0.05) whereas anti-CD3~inducedIL-10 was significantly inhibited by CI—A
`only (p < 0.05). No IL-IZ, IL-IO, or TNF-a was detected in cultures with
`or without added compounds in the absence of LPS or anti-CD3 (data not
`shown).
`
`were much more potent TNF—a inhibitors than the parent drug
`thalidomide,
`they dilfered in the slope of their dose-response
`curves as well as in the modulation of other monocyte and lym-
`phocyte cytokines. Members of one class of compounds, referred
`to here as class I, were broad inhibitors of the LPS-induced proin-
`flammatory monocyte cytokines TNF—oz , IL—IB, IL-6, and IL—12
`while potently augmenting the secretion of the antiinflammatory
`cytokine IL—lO. Class II compounds, on the other hand, i