Immunochemical characterization of antigenic domains on human IL-2: Spatially distinct epitopes are associated with binding to the p55 and p70 subunits of IL-2 receptor

0161-5890~92$5.00 + 0.00 Pergamon press plc

molecular l~~ffno~ogy. Vol. 29, No. I, pp. i19-130, 1992 Printed in Great Britain.

IMMUNOCHEMICAL CHARACTERIZATION OF ANTIGENIC DOMAINS ON HUMAN IL-2: SPATIALLY DISTINCT EPITOPES ARE ASSOCIATED WITH BINDING TO THE p55 AND ~70 SUBUNITS OF IL-2 RECEPTOR* ANGELITA REBOLLO,-~$ D~NAT DE GROOTE& MARC BAUDRIHAY,$ JACQUESTH~~zE? and DRAGANA LJ. JANKoVICt I( tUniti! d’ImmunogCnttique

Cellulaire, Institut Pasteur, 25 Rue du Dr Roux, 75015 Paris, France and $Medgenix Group, 6220 Fleurus, Belgium

(First receined 24 December 1990; accepted in revised form 3 April 1991)

Abstract-We have isolated and characterized 8 mAb against human rIL-2. All recognize nonglycosylated rIL-2 in liquid phase with similar affinities (& N 1 nM). Based on the epitopes of the IL-2 molecule that they recognize and their pattern of reactivity against glycosylated and nonglycosylated IL-2, they have been classified into four groups. The first group of anti-IL-2 mAb (2C4, 19Bll and 12C2) inhibits IL-2 binding to p70 IL-2R, while the second one (16F11, 18El and 2A4) prevents its binding to ~55 IL-2R. These two groups neutralize IL-2 activity in a T cell proliferation assay equally well, due to their similar inhibition of IL-2 binding to high affinity IL-2R. Two mAb, 3H9 and 17F4, recognize separate epitopes on IL-2 molecule, are poor inhibitors of IL-2 binding, and they are inefficient in the neutralization of its biological activity; they have been assigned to the third and fourth groups, respectively. These results show that mAb from the first and second group recognize two epitopes of the human IL-2 molecule which probably overlap the p70 IL-2R and ~55 IL-2R binding sites, respectively. In addition, these areas together form the high affinity IL-2R binding site. The two mAb from the third and fourth group recognized epitopes of IL-2 not directly involved in IL-2 binding to its receptor. All eight mAb anti-human IL-2 recognize murine IL-2 and with the exception of one, 17F4 mAb are also able to neutralize it in a T cell proliferation assay. The relationship between the structure and the function of the IL-2 molecule is discussed.

INTRODUCTION

The lymphokine IL-2 plays a critical role in the proliferative expansion of activated T cells (Smith, 1988). In addition, it has an immunomodulatory effect on a variety of lymphocytes, including T and B cells, NK and l~phokine-activated killer cells (Waldmann, 1989). The biological effects of IL-2 are mediated through specific interaction with cell-surface receptors present on the target cells. IL-2R are heterogeneous in terms of their biochemical and binding characteristics. The highaffinity IL-2R (& = 10 PM) is a complex of at least two distinct polypeptides of M, 55 kDa (~55 IL-2R) and M, 70-75 kDa (~70 IL-2R) (Sharon et al., 1986; Tsudo et al., 1986). Both the ~55 and p70 subunits of IL-2R can bind IL-2 independently with low (& = 10 nM) (Robb et al., 1984a; Leonard et al., 1984) and intermediate affinity *This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale (INSERM), the Association de la Recherche sur le Cancer and the Centre

National de la Recherche Scientifique (CNRS). $Supported by a grant from the Spanish Government. /Author to whom correspondence should be addressed at: Dragana Lj. Jankovic, Unite d’ImmunogCnttique Cellulaire, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris cedex 15, France. Abbreviations: ~55 IL-2R, subunit of IL-2R of M, 55 kDa; p70 IL-2R, subunit of IL-2R of M, 7&75 kDa.

(& = 1 nM) (Tsudo et aE., 1989; Hatakeyama et al., 1989), respectively. There is evidence for a more complex subunit structure for high affinity IL-2R that, besides ~55 and p70 IL-2R, involves non-IL-2 binding proteins such as ICAM- (Burton et al., 1990), MHC class I molecules (Sharon et aL., 1988) and an associated molecule, called p64 (Takeshita et al., 1990). In addition, recent data suggest that at least a portion of mouse p70 IL-2R appears to exist as a disulfide-linked heterodimer with a protein of M, 22 kDa (Saragovi and Malek, 1990) and/or is non-covalently associated with another protein of M, 100 kDa (Sharon et al., 1990; Colamonici et al., 1990). The three-dimensional structure of human IL-2 has been solved to 3.0 A resolution. IL-2 is an alpha-helical protein with no segments of beta-structure (Brandhuber et al., 1987). It is composed of 6 helical segments: A, B-B’, C, D, E and F that are separated by extended loops. The core of the molecule is made of anti-parallel alpha-helices B-B’, C, D and F linked by a disulfide bond between cysteine residues 58 and 105. Disruption of this intrachain disulfide bridge results in complete loss of IL-2 biological activity (Liang et al., 1986). Various approaches have been used to identify domains on the IL-2 molecule that are responsible for its interaction with the receptor. Site-directed mutagenesis has provided considerable data about functionally important areas of IL-2. However, the results obtained cannot always distinguish mutations which affect folding

and conformation from those which are in the active lymphocyte/NK cell line YT clone 2C2 (UT-2C2; site of the molecule. These studies have shown that Yodoi et al., 1985; Teshigawara et al., 1987) and IL-2.. N-terminal residues l-20 as well as C-terminal residues dependent clone TT9 (Diu et al., 1987) were used in the 121~-133 are required for full biological activity of the present study+ All cultures were performed in complete IL-2 molecule (Iu et al., 1987: Landgraf et al,, 1989). medium composed of RPMI 1690 (Bioproducts, WalkExtensive mutagenesis of the internal region between ersville, ME) supplemented with ~-glutamine (10m3 M), residues 30 and 60 did not identity additional amino HEPES ( 10e2 M), peniciIlin (100 U/ml>. streptomycin acids that were crucial for maintaining its activity. (100 tg/ml), 2-ME (5 x IO-‘M) and 5 or 10% fetal Elucidation of the subunit structure of the high-affinity calf serum (Flow Laboratories, Irvine, Scotland) for IL-2R complex has recently allowed a more detailed CTLL and YT-2C2 cells, respectively. The CTLL-2 analysis. Although various mutations at positions l-20 line and clone TT9 were maintained in complete and 121-133 of the IL-2 molecule lead to loss if IL-2 medium supplemented with SN from Con A-activated binding to both p55 IL-2R and p7O IL-ZR, replacement spleen cells from Fischer-344 rats and human rIL-2, of asparagine at position 20 in helix A by lysine gives rise respectively. to a biologically inactive protein that is unable to interReagents and ant ibodies act with high-affinity and p7O IL-2R, but that retains Human non-glycosylated rIL-2 was a generous gift of binding to ~55 IL-2R (Collins et al., 1988). This result shows that the N-terminal region of the IL-2 molecule Roussel Uclaf (Romainville, France). Human glycosylated rlL-2 was kindIy provided by Sanofi (Castanetis involved in binding to p7O TL-2R, while the area of Tolosan, France). SN from Con A-stimulated spleen interaction with the ~5.5 IL-2R remains unknown. cells from DBAJ2 mice was used as a source of native Another approach to the study of structure-function murine IL-2. relationship for the IL-2 molecule is based on the use of Soluble human ~55 IL-2R was obtained from T specific antibodies, with the advantage that the intact Cell Sciences (Cambridge, MA). Murine mAb 33.B3.1, IL-2 molecule can be analysed instead of peptides or directed against an epitope idemical or very close to the mutants that might have lost their native conformation. IL-2 binding site on the ~55 IL-2R molecule (Olive et al.. Many mAb against human IL-2 have been described 1986). was kindly provided by Dr Y. Jacques (INSERM (Gillis and Henney, 1981; Stadler et al., 1982; Robb U 211, Names, France). Rat mAb S4B6-I, specific for ei al., 1983; Smith et nl., 1983; Cousin rf al., 1985; Ohike murine IL-2 (Zurawski er al., 1986), was provided by Dr et nl., 1986; Brandt et ai., 1986; Ide et al., 1987), but their T. R. Mosmann (DNAX, Palo Alto, CA). effects on IL-2 binding to IL-2R were not analyzed, except for the ones described by Kuo and Robb (1986) Isolation and characterizatiolz of anti human IL-2 wlAb and Robb et al. (1987). Using antibodies directed against Female Balb/c mice, 8-12 weeks of age, were immudifferent areas of the IL-2 molecule, Kuo and Robb nized 5-7 times with 5 ,ug of human non-glycosylated (1986) have shown that residues 8-25 and 33-54 are rIL-2 at intervals of two weeks. rIL-2 was mixed with involved in high-affinity receptor interactions. MoreCFA or IFA for the first and second immunizations, over, it was shown that residues 8-2.5, which include respectively, and with PBS for the subsequent immunizhelix A, are involved in binding to p7O IL-2R (Robb et ations. Three days before fusion, the mice was boosted al., 1987), in agreement with the data from site-directed with an rIL-2 i.v. injection. Splenocytes were fused with mutagenesis. On the other hand, mAb that interact with the NSO myeloma using a modification of the procedure the region defined by residues 33-54 (helix B-B’) affect described by Galfre and Milstein (Galfre and Milstein, IL-2 binding to ~55 IL-2R and p70 IL-2R equally well 1981). (Robb et al., 1987). No antibodies inhibiting binding to Screening for anti-IL-2 activity was carried out by only p55 IL-2R have been described. immunoprecipitation. Culture supernatants were incuTo attempt to identify regions of IL-2 involved in bated with 1 ng of “‘I-IL-2 ~Amersham, U.K.) in 200 ~1 interactions with its receptors we have isolated and of K2HPUI/KH,P0, buffer supplemented with 0.1% characterized various murine mAb directed against BSA, After overnight incubation at 4°C 100~1 of human non-glycosylated rIL-2 and analysed their effect donkey anti-mouse immunoglobulins coated to cellulose on IL-2 binding to isolated ~55 IL-2R and p70 IL-2R, (IDS, Washington, U.K.) were added. The mixture was and to high-affinity receptor complex. In addition, the incubated for 30 min at room temp and then 1 ml of reactivity against different forms of human, as well as distilled water + 0.1% Tween 20 were added. Samples murine, IL-2 was tested. Using the anti-IL-2 mAb were centrifuged at 2,000 rpm for 5 min. Supernatants described in this paper, we were able to show that two were discarded and pellets were counted in a y-counter. separate regions of the IL-2 molecule are involved in The affinities of mAb for IL-2 were also determined by binding to p70 IL-2R and to ~55 IL-2R. immunoprecipitation with the exception that purified anti-IL-2 mAb at a constant concn were incubated with MATERIALS AND METHODS various doses (0.01-3 ng) of ‘251-IL-2. Non-specific bindCell lines and culture conditions ing was estimated in the absence of anti-IL-2 mAb. The The IL-Zdependent murine T cell line CTLL-2 calculated values for dissociation constants (&) were (Baker et al., 1979) and the human large granular derived by Scatchard plot analysis of binding cumes for

121

Monoc~onaI antibodies against human IL-2

each mAb. Only data which gave correlation coefficients > 0.95 were considered. In addition, the reactivity of anti-IL-2 mAb was tested by ELISA. Plates were coated with 10 ng of human non-glycosylated or glycoslyated rIL-2 in K,HPO$ KH,PO, 0.1 M pH 8 buffer. After washing and saturation, several dilutions of anti-IL-2 mAb were added. Bound anti-IL-2 mAb were detected by the p-galactosidase-conjugated rat anti-mouse K chain mAb 226 (kindly given by Dr M. Scharff, A. Einstein University, New York). Isotypes of anti-IL-2 mAb were determined using the kit supplied by Miles Scientific (Naperville, IL).

ELISA was used to test the reactivity of anti-IL-2 mAb against murine IL-2. Wells were coated with 0.25 pg of the rat anti-murine IL-2 mAb S4B6-1 in K,HPO.JKH,PO, 0.1 M pH 8 buffer. After 1 hr at 37°C wells were washed and saturated with BSA, followed by addition of SN from Con-A activated murine spleen cells (25% v~v). After incubation, wells were washed and several dilutions of anti-IL-2 mAb were added for an additional incubation, Wells were washed followed by the addition of a p-galactosidase-conjugated rat antimouse ICchain mAb 226. After 1 hr at 37°C substrate (p-nitrophenyl-/3-D-galactopyranoside) was added. The reaction was stopped by adding Na,CO, and wells were read in a spectrophotometer. sandwich RIA Labeling of purified anti-IL-2 mAb with Na’*‘I (Amersham, U.K.) was performed by the chloramine-T method (Bolton and Hunter, 1986). The sandwich assay was performed as follows: flexible 96-wells microtiter plates (Dynatech, Alexandria, VA) were coated with each anti-IL-2 mAb at a concn of 0.25 pg/well. After washing and saturation, serial dilutions of non-glycosylated rIL-2 were added to the wells. After 1 hr incubation at 37°C excess IL-2 was washed and a saturating concn of a given ?-anti-IL-2 mAb (0.03 pg/well) was added to the wells. Following incubation for 1 hr at 37°C and washing, individual wells were counted to determine the amount of bound radioactivity. inhibition ofIL-2 b~~d~~gto p 70 IL-2R and high -a..nity IL-2R IL-2 binding to p70 IL-2R or high-affinity IL-2R was measured using YT-2C2 or TT9 cells, respectively, and was performed according to the method of Robb et al. (1984a). Serial dilutions of anti-IL-2 or control mAb were incubated with 1 ng or 0.15 ng of human nonglycosylated ‘*’I- IL-2 when YT-2C2 or TT9 cells were used, respectively. These doses of “SI-IL-2 give 30% of maximum binding to the corresponding cells. Anti-p55 IL-2R mAb (33B3.1; 0.2 PM) added at saturating concn did not affect IL-2 binding to the YT-2C2 cells. After 1 hr of incubation at room temp, 1 x lo6 cells were added to give a total reaction volume of 100 ~1. Following 60 min incubation at 4°C the reaction was stopped

and the cells were centrifugated. Cell bound 12$I-IL-2was separated from free ‘?-IL-2 by centrifugation over a cushion consisting of 84% silicone and 16% Vaseline. The tips of the tubes containing cell pellets were cut and counted in a y-counter. Binding of ‘251-IL-2measured in the presence of control mAb was used as a positive control, while non-s~cific binding was estimated by adding a 200-fold excess of unlabeled IL-2. Inhibition of binding of anti-p55 IL-2R mAb 3383.1 to soluble ~55 IL-2R Soluble ~55 IL-2R was coated overnight at 4°C on flexible 96-well round bottom microtiter plates in 50 ~1 volumes and the plates were saturated with BSA. Coated ~55 IL-2R was revealed by anti-p55 IL-2R mAb, ‘25133B3.1, after 2 hr incubation at 37°C. Using various concns of 33B3.1 mAb we estimated that 0.03 pg/well of this mAb gives 40% of maximum binding. The suboptimal concn of IL-2, inhibiting binding of 0.03 pg/ well ‘2s1-33B3.1 by 50%, was determined to be 0.15 pg in a direct inhibition assay. To test the effect of anti-IL-2 mAb on IL-2 binding to ~55 IL-2R we preincubated 0.15 c(g of IL-2 with increasing concns of each anti-IL-2 mAb for 2 hr at 37°C. These test samples were then transferred to ~55 IL-2R coated plates. After 2 hr at 37°C 0.03 pgg/well of 12’1-33B3.1mAb was added to each well followed by an additional 2 hr incubation at 37°C. The final volume added to ~55 IL-2R coated wells was kept constant (50 ~1) throughout. The plates were washed and cut into single wells. Radioactivity corresponding to bound ‘2sI-33B3.1 was determined using a y-counter. Prolferation assays Various concns of anti-IL-2 mAb were incubated with doses of IL-2 given half-maximal rH]dThd incorporation followed by addition of CTLL-2 cells (lo4 cells/ well) in a final volume of 200 yl. Corresponding doses of human non-glycosylated or glycosylated rIL-2 and murine native IL-2 were previously determined. Cultures were pulsed with 0.5 pCi/well of [3H]dTdh after 24 hr of incubation and 16 hr later the cells were harvested by using a Titertek harvester and radioactivity incorporated into DNA was measured in a ~-scintillation counter. RESULTS Reactivity of mAb against human IL-2 We have isolated and characterized 8 different mAb against non-glycosylated human rIL-2 (Table 1). They were identified by liquid phase immunoprecipitation using “‘I-IL-2 and anti-mouse immunoglobulins coated to cellulose. The dissociation constant of the mAb/ protein complex was measured using the same method. The results were analyzed by Scatchard plot and the equilibrium dissociation constant was obtained by linear regression. All of the anti-IL-2 mAb have similar affinities since their I(d ranged from 1.1 nM to 3.4 nM (Table 1). All anti-IL-2 mAb are of the IgGl isotype, except mAb 16Fll whose isotype is IgG2a.

122

A. REBOLLOet nl. Table I. Characteristics

of the anti-IL-2 mAb used in this study

Liquid phase” RIA Antibody

___I (X4) 2(19Bll) 3 (12C2) 4 (16F1 I) 5 (18El) 6 (2A4) 7 (3H9) 8 (17F4) 9 Controld

Isotype’

IgGl IgGl IgG 1 IgG2a IgGl IgGl IgGl IgGl IgGt

Kd (nM)

- -_

1.3 1.1 2.1 2.0 I.6 3.4 2.9 1.5 -

Solid phase” ELISA Non-glycosylated Glycosylated

IL-2 ._~ -

Symbol

+ + -

-

+ + -

+ + -

0 0 n L1 a A 0 CI

IL-2 ~~ ~____ _.._ +

-

0

“All mAb recognize human non-glycosylated IL-2 in liquid phase. K, was estimated by Scatchard plot computer analysis of equilibrium binding. ‘Human non-glycosylated or glycosylated IL-2 was coated on the ELISA plate and bound anti-IL-2 mAb were detected with b-galactosidase-conjugated rat antimouse K chain mAb. ‘Isotype of anti-IL-2 mAb was tested in direct ELISA. *‘Mouse anti-rat K chain mAb (IgGl) was used through the experiments as a control mAb.

The anti-IL-2 mAb can be classified into three groups on the basis of their reactivity against nonglycosylated or glycosylated human IL-2 coated on ELISA plates (Table 1). The first group of anti-IL-2 mAb (2C4, 19Bll and 1X2) reacted in the ELISA test

only with non-glycosylated human IL-2. The second group of anti-IL-2 mAb (16Fl1, 18El and 2A4) did not recognize either non-glycosylated or glycosylated human IL-2 in the solid phase assay. Two anti-IL-2 mAb, 3H9 and 17F4, form the third group, since they recognized both types of human IL-2 equally well in ELISA. As we will discuss below, these two mAb can be further separated into two independent groups, composed of one anti-IL-2 mAb, according to the epitopes they recognize. Relation between epitopes recognized by d$erent

mAh

To evaluate the number of epitopes recognized by this panel of anti-IL-2 mAb, we assayed pairs of antibodies for their ability to simultaneously bind to non-glycosylated human IL-2 in the RIA sandwich assay. Plates were coated with each anti-IL-2 mAb, incubated with IL-2, and different concns of a given radiolabeled mAb 2 lz5I-3H9 12 D were added. 12$1-1 2C2 mAb could recognize the IL-2 molecule when the coated mAb was 16F11, 18E1, 2A4, 3H9 or 17F4, but not when the coated mAb was 2C4, 19Bll or 12C2 (Fig. IA). This together with results in Table 1, show that all three anti-IL-2 mAb in the first group (2C4, 19Bl I and 12C2) recognize the same epitope, or closely related ones, distinct from the epitopes recognized by the other mAb. When a radiolabeled mAb from second group, ISEI, was similarly tested, it was found to recognize IL-2 if the coated mAb was from the first group, or 3H9 (Fig. t B). Fig. 1. Simultaneous binding of two anti-IL-2 mAb to IL-2. However, when the coated mAb was 3H9, radiolabeled RIA plates were coated with 025,~g/well each of anti-IL-2 18E1 recognized IL-2 less efficiently than when the mAb 2C4, or 19311, or 12C2 (0); 16Fl1, or 18E1, or coated mAb came from the first group. mAb ‘“‘I-18El 2A4 (A); 3H9 (A) or 17F4 (a) followed by addition of could not bind to IL-2 if the coated mAb was from serial dilutions of non-glycosylated rIL-2. After incubation, 0.03 pg of ‘**I-12C2 (panel A), iZSI-18El (panel B), 12’1-3H9 the same group (16F11, 2A4 and 18El) indicating, together with similar behaviour in ELISA (Table l), that (panel C) or iZSI-17F4 (panel D) was added. Rest&s are these mAb recognize the same epitope, or closely related represented as cpm of bound radiolabeled anti-IL-2 mAb for ones. Surprisingly, mAb 18El did not recognize ?L-2 each dilution of rIL-2.

Monoclonal

123

antibodies against human IL-2

Antibody

concentration

(pghl)

Fig. 2. Effect of anti-IL-2 mAb on binding of rzsI-IL-2 to p70 IL-2R. Various concns of anti-IL-2 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ with 1 ng of L251-IL-2in 50 ~1 followed by the addition of 50 ~1 containing 1 x lo6 YT-2C2 cells. After incubation, cell-bound and free ‘251-IL-2were separated as described in Materials and Methods. The results are expressed as per cent of inhibition of binding of ‘251-IL-2to YT-2C2 cells in the presence of anti-IL-2 mAb. ‘251-IL-2 bound to YT-2C2 cells in the presence of control mAb gave 5,152 + 230 cpm. Background cpm were estimated in the presence of 200-fold molar excess of non-labeled IL-2 and were 432 + 31 cpm. cpm in the presence of anti-IL-2

mAb - background

cpm in the presence of control mAb -background

bound to mAb 17F4, even though they do not belong to the same group (Table 1). Radiolabeled mAb 3H9 could recognize the IL-2 molecule independently of the coated mAb, except when the coated mAb was 3H9 (Fig. 1C). A very similar situation was observed for mAb 17F4 (Fig. ID). Since mAb 3H9 can react with IL-2 bound to mAb 17F4, and vice versa, the two mAb recognize distinct epitopes on the IL-2 molecule, in spite of a similar ELISA reactivity pattern (Table 1). In contrast to the absence of recognition by mAb 18El of IL-2 bound to mAb 17F4, the latter reacts well with IL-2 bound to mAb 18El (Fig. 1D). This discrepancy suggests that IL-2 bound to 17F45 has an altered conformation which is no longer recognized by 18E1, but that the reverse effect does not occur. These results were confirmed by cross-inhibition experiments with ‘251-labeled mAb from the first, third and fourth groups that recognize non-glycosylated IL-2 in the solid phase assay (data not shown). Therefore, the 8 anti-IL-2 mAb can be classified into four groups which recognize at least four different epitopes on the IL-2 molecule. Effect of mAb on binding of IL-2

to ~70 IL2R

To further characterize the four groups of anti-IL-2 mAb, we studied their ability to inhibit the binding of IL-2 to p70 IL-2R. Figure 2 shows the ability of each mAb studied to inhibit IL-2 binding to the YT-2C2 cell line, which expresses p70 IL-2R only (Teshigawara et al., 1987). mAb belonging to the first group (2C4, 19Bll and 12C2) were the only mAb able to completely inhibit ‘*‘I-IL-2 binding to YT-2C2 cells with 50% inhibition

cpm cpm >

x 100.

requiring only 0.01 pgg/ml of mAb. To obtain the same level of inhibition, 1 pg/ml of mAb 17F4 or 3H9 and 10 pg/ml of mAb from the second group (16F11, 18El and 2A4) were needed. While mAb from the second, third and fourth group only partially inhibits IL-2 binding to p70 IL-2R even at lOO-fold mAb molar excess, almost complete inhibition is achieved using an equimolar concn of mAb from the first group. These results strongly suggest that the later anti-IL-2 mAb recognize an area of IL-2 directly involved in binding to p70 IL-2R. E#ect

of mAb on binding of IL-2

to ~55 IL-2R

The anti-IL-2 mAb obtained were tested for their ability to inhibit IL-2 binding to ~55 IL-2R. Since high concns of IL-2 are required to saturate ~55 IL-2R, an indirect assay was employed. Soluble ~55 IL-2R was coated on RIA plates and detected by 12’I-33B3.1 mAb that competes with IL-2 for its binding site on the ~55 IL-2R molecule (Olive et al., 1986). Initially we determined the concn of non-glycosylated IL-2 that inhibits binding of a given concn of ‘*‘I-33B3.1 mAb by 50%, which corresponds to 40% of maximum binding to soluble ~55 IL-2R. IL-2 at this concn was then preincubated with various concns of each anti-IL-2 mAb. The effect of the anti-IL-2 mAb on binding of IL-2 to ~55 IL-2R was measured by increased binding of ‘251-33B3.1 mAb. Figure 3 shows that only mAb from the second group (16F11, 18El and 2A4) inhibited binding of IL-2 to ~55 IL-2R, permitting more “‘1-33B3.1. mAb to bind to ~55 IL-2R. mAb from the first, third or fourth group did not affect binding of IL-2 to ~55 IL-2R at any concentration tested, since the binding of ‘2SI-33B3.1

124

A. REBOLLO et ui.

shown). These results show that mAb from the second group recognize a region of IL-2 important in binding to ~55 IL-2R.

We further analyzed which epitopes recognized by our anti-IL-2 mAb are involved in IL-2 binding to high-affinity IL-2R complex. Inhibition experiments were performed using human clone TT9 that express high-affinity IL-2R. The concn of IL-2 used was sufTicient to saturate 50% of the high-affinity I , 1 I I I t , IL-2R complexes. The results are shown in Fig. 4. 0.6 0.15 2.5 10 Anti-IL-2 mAb from the first (2C4, 19BfI and Antibody concentration (pg/welt) 12C2) and second (16Fl1, 18El and 2A4) groups Fig. 3. Inhibition of binding of anti-p% IL-2R mAb ‘251- were able to completely inhibit binding of IL-2 to 33B3.1 to soluble ~55 IL-2R. A dose of human non-glycosyhigh-affinity IL-ZR, while with highest concn of lated rIL-2 (0.15,~g) that partially inhibited binding of mAb from the third to fourth groups we could only 12%33B3.1 mAb to soluble ~5.5 IL-2R was incubated with achieve 40% inhibition. To inhibit “‘I-IL-2 binding different dilutions of anti-IL-2 mAb 2C4, or 19Bl1, or 12C2 to high affinity IL-2R by 50% we needed the same (0). 16Fl1, or 18E1, or 2A4 (a); 3H9 (A) or 17F4 (0). After quantities of mAb from the first and second group incubation, the mixture was added to RIA plates coated with that inhibit IL-2 binding to p70 and ~55 IL-2R, respectsoluble ~55 IL-2R followed by addition of ‘251-33B3.1mAb. ively. Anti-IL-2 mAb from the third and fourth The effect of anti-IL-2 mAb was measured as increased binding groups, which are poor inhibitors of IL-2 binding to of 1251-33B3.1mAb to coated pS5 IL-2R. The binding of p70 IL-2R, and which do not affect IL-2 binding ‘?-33B3.1 mAb in the presence of 0.15 pg of IL-2, preincuto ~55 IL-2R, were also poor inhibitors of IL-2 binding bated with control mAb, was 5,431 & 106 cpm and in the absence of IL-2 was 13,051 + 200 cpm. to high-affinity receptor. These results suggest that the epitopes recognized by mAb 17F4 and 3H9 are separate from the receptor-binding site of IL-2. Very mAb was unchanged. Identical results were obtained similar results were obtained using the murine IL-2 when we analyzed binding to ~55 IL-2R on fibroblast cells that express only human p55 IL-2R (data not dependent T cell line CTLL-2 (data not shown).

091

Antibody

concentration

10

1000

(@g/ml)

Fig. 4. Inhibition of binding of “‘I-IL-2 to high-affinity IL-2R. Various dilutions of anti-IL-2 mAb 2C4, or 19Bl1, or 12C2 (0); 16Fl1, or 18E1, or 2A4 (A); 3H9 (A) or 17F4 (0) were incubated with 0.15 ng of ‘*%IL-2 in 50 p 1 followed by the addition of 50 p 1containing 1 x lo6 TT9 cells. After incubation, cell bound and free J2sI-IL-2 were expressed as described in Materials and Methods. The results are expressed as per cent inhibition of binding of “‘I-IL-2 to TT9 cells in the presence of anti-IL-2 mAb. ‘2sI-IL-2 bound to TT9 cells in the presence of control mAb gave 1,826 k 102 cpm, and background cpm, estimated in the presence of 200-fold excess of non-labeled IL-2, were 176 + 23 cpm. cpm in the presence of anti-IL-2 mAb - background cp x loo. cpm in the presence of control mAb - background cpm >

~onoclonal

antibodies against human IL-2

Antibody

concentration

125

(pglwell)

Fig. 5. Effect of anti-IL-2 mAb on the proliferative response of CTLL-2 in the presence of human rIL-2. Different concns of anti-IL-2 mAb 2C4, or 19B11, or 12C2 (0); 16FI1, or 18E1, or 2A4 (A); 3H9 (A) or 17F4 (0) were incubated with IOpg of non-glycosylated (panel A) or glycosylated (panel I$) human rIL-2, followed by the addition of I x IO4 CTLL-2 ~Ils/weIl in a final volume of 200 pl. After 24 h incubation, cultures were pulsed for I6 h with t3H]dThd. The results are given as per cent of inhibition of proliferation in the presence of mAb calculated as: cpm in the presence of anti-IL-2 mAb - background cpm cpm in the presence of control mAb -background cpm

x 100.

The proliferation in the presence of control mAb was 38,864 cpm and 37,321 cpm for nonglycosylated and glycosylated IL-2, respectively. The medium control was 365 cpm and SD did not exceed 10%.

E#ect of mAb on the proliferation of CTLL-2 cells in the presence of human rIL-2

efficiency in inhibition of proliferation in the presence of glycosylated human IL-2 in comparison to non-glycosyl-

To more fully characterize our anti-IL-2 mAb, neutralization assays were performed. Identical results were obtained using the murine T cell line CTLL-2 (data not shown) and human T cell clone TT9 (data not shown). The effect of anti-IL-2 mAb on the proliferative response of CTLL-2 cells in the presence of human non-glycosyiated IL-2 is shown in Fig. 5A. As expected from binding experiments, mAb from the first and second groups inhibited IL-Zmediated proliferation equally well. To reach 50% inhibition with mAb from the first and second groups, we needed lo- and 30-fold lower concns than with mAb from the third and fourth groups, respectively. Figure 5B shows the effect of anti-IL-2 mAb on CTLL-2 proliferation in response to glycosylated IL-2. The second group of mAb are the best inhibitors of proliferation mediated by glycosylated IL-2 and these mAb give profiles comparable to that shown in panel A with non-glycosylated IL-2. In contrast, anti-IL-2 mAb included in the first group do not affect the proliferative response to glycosylated IL-2, presumably due to lack of recognition. Anti-IL-2 mAb from the third and fourth groups are poor inhibitors of proliferation driven by glycosylated IL-2 and do not completely neutralize IL-2 activity even at concentration of 10 hgg/well of antibody, an approximate 6,600-fold excess of mAb molecules to IL-2. In the case of mAb from the fourth group, we observed a decreased

ated form. These results showed that mAb that could efficiently interfere at the level of IL-Zreceptor interaction were the best in neutralizing its biological activity. recognition of murine IL-2 by anti-human IL-2 mAb in ELISA and proliferation assay We also tested the ability of the four groups of mAb to recognize murine IL-2. ELISA plates were coated with the rat anti-mouse IL-2 mAb S4B6-1, followed by addition of native murine IL-2. After incubation, different concns of anti-IL-2 mAb were added. All the mAb recognized murine IL-2 (Fig. 6). The effect of anti-IL-2 mAb on the proliferation of CTLL-2 cells induced by murine IL-2 was also analysed. mAb from the first, second and third group could completely neutralize murine IL-2 with comparable efficiency (Fig. 7). mAb from the fourth group (17F4) marginally affected proliferation of CTLL-2 cells in the presence of murine IL-2 even at a l,OOO-fold molar excess. Identical results were obtained when murine rIL-2 was tested (data not shown). These results show that all eight anti-human IL-2 mAb recognize murine IL-2, suggesting a high degree of similarity to human IL-2. Areas recognized by mAb from the first, second and third group, but not fourth group, are involved in the biological activity of murine IL-2, probably in its binding to high-affinity IL-2R.

I26

4

Antibody

16

concentration

60

250

(nglwell)

Fig. 6. Recognition of murk IL-2 by anti-IL-2 mAb in ELISA. Plates were coated with 0.25 @g/wellof rat anti-mouse IL-2 mAb (S4B6-1) followed by the addition of SN from ConA-stimulated murine spleen cells. After incubation, several dilutions of anti-IL-2 mAb 2C4, or 19B11, or 12C2 (0); 16FI1, or I8E1, or 2A4 (A); 3H9 (A) or 17F4 (0) were added. Bound anti-IL-2 mAb were detected by a fl-galactosidase-conjugate rat anti-mouse K chain mAb. DISCUSSION

We have described eight murine mAb against human IL-2, that were identified by immunoprecipitation of non-glycosylated rIL-2. All the anti-IL-2 mAb have similar a~nities for rIL-2, and they have been classified into four groups according to the IL-2 epitopes they recognize. The first group of anti-IL-2 mAb efficiently inhibits IL-2 binding to p70 IL-2R, while the second one

Antibody

prevents its binding to ~55 IL-2R. These two groups equally well inhibit IL-2 binding to high-affinity IL-2R, and also neutralize IL-2 activity in a T cell proliferation assay. These results suggest that mAb from the first and second groups recognize different, spatially distinct areas of the IL-2 molecule that correspond to the ~70 and p.55 IL-2R binding sites, respectively. These two areas together form the high-affinity IL-2R binding site. The third and fourth groups of anti-IL-2 mAb recognize epitopes that are not close to IL-2R binding sites, since they do not completely inhibit IL-2 binding nor neutralize its biological activity. All four groups of anti-human IL-2 mAb recognize murine IL-2. In addition, the first three groups of anti-IL-2 mAb can neutralize murine IL-2, thereby showing similarities, but also differences, between the active sites of the two IL-2 molecules. Human IL-2 is a polypeptide of 133 amino acids (Taniguchi et al., 1983). Biochemical characterization of its natural form showed that the molecule is heterogeneous with respect to size and charge (Robb and Smith, 1981; Taniguchi et al., 1983). Variations in glycosylation are the source of this heterogeneity since IL-2 is the product of a single mRNA species from a single-copy gene. Post-translational modification of IL-2 molecule is restricted to different 0-glycosylation of threonine in position 3 (Robb et al., 1984b). In agreement with the report by Brandt et al. (1986), we show that mAb elicited against non-glycosyiated IL-2 can react with glycosylated IL-2. This result is in contrast to the results from other studies on anti-IL-2 mAb which were reported to be mainly reactive against the immunizing form of IL-2 (Cousin et al., 1985). While the second,

concentration

(pglwell)

Fig. 7. Effect of anti-IL-2 mAb on the proliferative response of CTLL-2 in the presence of murine IL-2. Serial dilutions of anti-IL-2 mAb 2C4, or 19Bl1, or 12C2 (0); 16F11, or 18E1, or 2A4 (A); 3H9 (A) or 1’7F4 (0) were incubated with SN from Mona-stimulated murine spleen cells (15% V./V) followed by the addition of 1 x IO4 CTLL-2 cells/well in a final volume 200,ul. After 24 hr incubation, cultures were pulsed with 13H]dThd for 16 hr. Cells were harvested and incorporated radioactivity was measured. The results are given as per cent of inhibition of proliferation in the presence of anti-IL-2 mAb. cpm in the presence of anti-IL-2 mAb - background cpm cpm in the presence of control mAb- background cpm The proliferative

x 100.

response of CTLL-2 cells in the presence of control mAb was 36,532 and the medium control was 420 cpm (SD < 10%).

Monoclonal antibodies against human IL-2 third and fourth group of anti-IL-2 mAb recognize both non-glycosylated and glycosylated IL-2, the first group of anti-IL-2 mAb recognizes only non-glycosylated IL-2. This difference in reactivity suggests that the epitope recognized by mAb from the first group is at the N-terminal region of the IL-2 molecule and is either absent or masked following glycosylation. The three-dimensional structural data suggest that N-terminal helix A (ll-19), helix B-B’ (33-56) and the peptide connecting them, as well as helix E (107-l 13) are likely potential receptor binding sites of the IL-2 molecule (Brandhuber et al., 1987). The anti-IL-2 mAb which recognize an epitope including residues 8-27 inhibit IL-2 binding to p70 IL-2R, and not to p55 IL-2R (Robb et al., 1987). Site-directed mutagenesis studies specifically point to the importance of arginine at position 20 for IL-2 binding to p70 IL-2R, but not ~55 IL-2R (Collins et al., 1988). The effect of our anti-IL-2 mAb on binding of IL-2 to ~55 IL-2R and p70 IL-2R was analysed. Anti-IL-2 mAb from the first group inhibit IL-2 binding to p70 IL-2R lOO- or l,OOO-fold more efficiently than the other mAb (Fig. 2) and do not at all affect IL-2 binding to ~55 IL-2R (Fig. 3). The different capacities of anti-IL-2 mAb to inhibit IL-2 binding to p70 IL-2R cannot be explained by differences in affinity since all of them have a similar dissociation constant (Table 1). It rather suggests that mAb of the first group recognize a region of the IL-2 molecule involved in binding to p70 IL-2R, which is likely to be the N-terminus, based on the difference in mAb reactivity against non-glycosylated and glycosylated forms of IL-2. The area of IL-2 recognized by mAb from the first group is not completely identical to the one seen by p70 IL-2R inasmuch as glycosylated IL-2 is recognized by its receptor and not by the mAb. In general, it is accepted that glycosylated and non glycosylated forms of IL-2 have the same affinity for high-affinity IL-2R complex (Robb et al., 19843); however these studies did not include direct competition binding studies using the p70 IL-2R subunit alone. Partial inhibition of IL-2 binding to p70 IL-2R by mAb from the third and fourth groups and from the second group in lOO- and l,OOO-fold molar excess, respectively, can be due to steric hindrance since an Ab has lo-times higher M, than IL-2. It is still possible, however, that other parts of the IL-2 molecule, besides the N-terminal region, are implicated in maintaining the p70 IL-2R binding site. Collins et al. (1988) showed that deletion of phenylalanine at position 124 prevents binding to p70 IL-2R and abolishes 60% of IL-2 binding to ~55 IL-2R. These results, together with the ones obtained by Landgraf et al. (1989) indicate that the C-terminal region of IL-2 is important for its full biological activity and its binding to high affinity IL-2R, but whether it is directly involved in binding to one of the IL-2R subunits, or instead plays a structural role in the conformation of IL-2, is not clear. An alternative, less likely, explanation for the partial inhibition of IL-2 binding to p70 IL-2R by the mAb from the third and fourth groups is that they inhibit binding of IL-2 to the

127

putative ~100 subunit of IL-2R that might be a partner of p70 IL-2R in formation intermediate affinity IL-2R (Colamonici et al., 1990). The ~55 IL-2R binding site on the human IL-2 molecule is not characterized. Various amino acid replacements affect IL-2 binding to ~55 IL-2R (Collins et al., 1988; Landgraf et al., 1989). However, among the X-2 mutants unable to bind ~55 IL-2R tested so ar, none retained binding to p70 IL-2R. Similarly, mAb that inhibit IL-2 binding to ~55 IL-2R also inhibit binding to p70 IL-2R with the same efficiency (Robb et al., 1987). In our tests the second group of anti-IL-2 mAb was the only one that inhibited IL-2 binding to ~55 IL-2R. At equimolar concns these mAb competely inhibited IL-2 interaction with ~55 IL-2R (Fig. 3) but only inhibited interaction with p70 IL-2R by 15% (Fig. 2). We have observed that mAb from the second group cannot react with IL-2 molecule previously bound to mAb from the fourth group, and only weakly with IL-2 bound to mAb from the third group. However, mAb from the third and fourth groups react well with IL-2 to which second group mAb have been bound. It is possible that interaction of IL-2 with mAb from the third and fourth groups induce some conformational changes in the IL-2 molecule, thereby preventing its reaction with mAb from the second group. This group of mAb recognize both non-glycosylated and glycosylated IL-2, but only in liquid phase. This indicates that the native conformation of IL-2 is required for recognition by these mAb and the same may be true for ~55 IL-2R. IL-2 binding to high affinity IL-2R complex is a composite of its binding to individual receptor subunits: with its rapid association constant it resembles ~55 IL-2R binding and its slow dissociation constant to p70 IL-2R binding (Lowenthal and Greene, 1987). The contact sites of IL-2 with high affinity IL-2R complex are at least the sum of contacts between IL-2 and individual chains. In our study, the anti-IL-2 mAb from the first and second groups could completely inhibit IL-2 binding to high-affinity IL-2R (Fig. 4). These two sets of mAb blocked IL-2 binding to either ~55 IL-2R or p70 IL-2R, but they equally well inhibited IL-2 binding to highaffinity IL-2R. While equimolar concns of mAb from the first and second group completely prevent IL-2 binding to ~55 IL-2R and p70 IL-2R, respectively, at lOO- and lOOO-fold molar excesses, they inhibit IL-2 binding to high-affinity IL-2R by 50% and 100% respectively, This difference in mAb concn requirement can be explained by differences in affinities for individual subunits versus the high affinity IL-2R complex. The ~55 IL-2R, p70 IL-2R and anti-IL-2 mAb have comparable affinities for IL-2 (K, = l-10 nM), while the affinity of high-affinity complex for IL-2 is l,OOO-fold higher affinity. Even at large excesses of mAb from the third and fourth groups only partial inhibition can be achieved, suggesting that binding of these mAb affects IL-2 binding, not by direct competition for the same binding site, but rather by steric hindrance. In addition, we have tested four other groups of anti-IL-2 mAb that did not affect IL-2 binding to ~55 IL-2R or p70 IL-2R, and did not find that they

128

A. REBULLO et ul.

could prevent high-affinity binding (data not shown). Therefore, we were not able to identify an additional high affinity IL-2 receptor binding area, other than the one involved in IL-2 binding to ~5.5 IL-2R and p70 IL-2R. The recently described putative ~100 subunit of IL-2R was shown not to be involved in IL-2 binding to high affinity IL-2R complex (Colamonici et ul., 1990). The inhibition of T cell proliferation by anti-IL-2 mAb in the presence of non-glycosylated IL-2 had a very same pattern to inhibition of IL-2 binding to highaffinity IL-2R (Fig. 5A). Similar results were obtained with glycosylated IL-2, with the exception of lack of inhibition with mAb from the first group which do not react with glycosylated IL-2 (Fig. SB, Table 1). Human IL-2 has the same, or higher, specific activity on murine as on human T cells (Roifman et al., 1985: Mosmann et al., 1987). High-affinity IL-2 binding tests, as well as T cell proliferation assays, were performed in parallel experiments on the murine CTLL-2 cell line and the human IL-2 dependent T cell clone TT9. The results were identical in both cases, indicating similarities in regions of the IL-2 molecule seen by human and murine high-affinity IL-2 receptors. Although human and murine IL-2 have 60% homology at the protein level (Yokota et al., 1985), murine IL-2 is IOO-fold more active on mouse than human cells (Mosmann et al., 1987). The main difference between the two is an insert of 12 consecutive glutamines in the N-terminal region of murine IL-2. The three-dimensional structure of murine IL-2 has not been determined. The first 26 amino acids of murine IL-2, including a poly-glutamine stretch are tolerant to change (Zurawski et al., 1986). The region immediately distal to the glutamine stretch (31-39) is critical to the activity of murine IL-2, and it has been suggested that this region is involved in binding to p70 IL-2R (Zurawski and Zurawski, 1988, 1989). Disruption of the region between 1222128 is known to affect ~55 IL-2R binding (Zurawski and Zurawski, 1989). By analogy to human IL-2, this area of murine IL-2 corresponds to helix E. The rat mAb S4B6-1 specifically recognizes and neutralizes murine but not human IL-2 (Zurawski et al., 1986). We have used this mAb, along with the observation that mouse IL-2 consists of a dimer, in performing the ELISA sandwich test which demonstrated reactivity of all eight anti-human IL-2 mAb towards murine IL-2. In addition, they recognize native and rIL-2 in solution equally well (Fig. 6 and data not shown). Cross-reactivity between anti-human IL-2 mAb and murine IL-2 has often been observed (Gillis and Henney, 1981; Stadler et al., 1982; Smith et al., 1983). The first, second and third groups of anti-IL-2 mAb completely inhibited the proliferation of CTLL-2 cells induced by murine IL-2, while the fourth group of mAb did not neutralize its activity (Fig. 7). The concns of mAb from the first and second groups required to neutralize murine IL-2 were comparable to those necessary for neutralizing human IL-2. Together these data suggest that three groups

the epitopes on murine

recognized by from the first IL-2 are likely involved in its

binding to high-affinity IL-2R. It is interesting to point out that while mAb 3H9 only partially neutralized human IL-2, it completely blocked murine IL-2. which implies a difference between the active sites of the two IL-2 molecules. This work has permitted a discrimination between areas of IL-2 involved in binding to the ~55 IL-2R and p70 IL-2R using anti-IL-2 mAb. Further characterization of the epitopes of IL-2 by these mAb will be required for a more complete identification of the regions of IL-2 responsible for interaction with its receptors and for its biological function. Acknowledgements-We thank Drs Y. Jacques and T. Mosmann for their generous gift of monoclonal antibodies 33B3.1 and S4B6, respectively. Thanks are also due to Sanofi and Roussel Uclaf for generous supply of rIL-2, and T Cell Sciences for Providing us with soluble ~55 IL2R. We are grateful to Dr J. Yodoi for YT-2C2 clone. We thank J. L. Moreau for computer design of the graphs, and A. Bas and S. Van Elstraete for expert secretarial assistance.

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Zurawski S. M. and Zurawski G. (1988) Identification of three critical regions within mouse interleukin 2 by fine structural deletion analysis. EMBO J. 7, 1061-1069. Zurawski S. M. and Zurawski G. (1989) Mouse interleukin-2 structure-function studies: substitutions in the first a-helix can specifically inactivate p70 receptor binding and mutations in the fifth a-helix can specifically inactivate ~55 receptor binding. EMBO J. 8, 2583-2590.