stRyefets| x1 (1988), M21 WZ Korean Biochem. J. (1988), Vol. 21, No. 2, pp. 127~133 Recombinant Human Interleukin-2: I]. Biological and Therapeutic Activities Hye-Lim Choit, Hye-Young Yun*, James E. Talmadge” and Kyung-Soo Hahm** Protein Chemistry Laboratory, Genetic Engineering Center, KAIST, P.O. Box 131, Cheong Ryang! Seoul, Kored! and Preclinical Screening Laboratory, National Cancer Institute, Frederick Cancer Research Facilities, Frederick, MD, U.S.A. (Received May 12, 1988) Abstract : Recombinant human interleukin-2 (rH IL-2, Ser'®*-rH IL-2) purified from E. coli was characterized its biological and therapeutic activities for the establishment of preclinical screening system and therapeutic applications. In vitro augmentation of natu ral killer (NK) cell activity and mixed lymphocyte reaction (MLR) assay were shown to be comparable with other rH IL-2 from different manufacturers and laboratories. An antiserum against rH IL-2 was obtained from chick, and characterized the reacticvity with rH IL-2 reconstituted from lyophilized state in relation to the stability and confor- mational changes of the recombinant protein. In addition, long term storage of rH IL-2 in lyophilized or solution was not shown to incur remarkable loss of MLR activities, and any appearance of either degradation or aggregation products when analyzed by SDS-PAGE, indicating the stability and suitability for therapeutic applications of rH IL-2, Human recombinant interleukin-2 (rH IL-2) has the appeal of immunotherapy, but clinical trials have generally been disappointing, with results that were inferior to those obtained in a variety of anim- al models (Terry et al, 1982; Mihibch et al, 1983; Oldham et al, 1983; Talmadge et al, 1985a,b). the positive results seen in experimental systems appear to be due to the initiation of therapy in nor mal animals with minimal tumor burden. This may also have been the origin of the disparity between the preclinical therapeutic activity of cytotoxic drugs and their clinical properties, Many preclinic- al immunotherapy studies have actually investi- gated immune prophylaxis, since experimental im munotherapy can be initiated prior to, or simul taneously with tumor implantation. Also, central to the identification of biological response modifiers (BRMs) useful in clinical oncology is the develop- “Reprint requests to Kyung-Soo Hahm. *This work was supported in part by the grant (F80010) from the Ministry of Science and Technology, Korea ment of the experimental protocols for the treatment of animals with preexistent, well-established metas- tasis, as well as investigations into the therapeutic modulation of the host immune response to primary autochthonous neoplasms and associated metasta: sis. To this end, many cancer research laboratories and pharmaceutical industries developed systems for screening drugs for antitumor activity. ‘The recombinant human IL-2 (KAIST rH IL-2) and a mutein (KAIST Ser'*°-rH IL-2) have been successfully cloned and over produced from E. coli (Han, 1987), and purified to an apparent homogeneity (Yun et al, preceding paper in this volume). The present study was therefore under taken in order to evaluate biological and therapeu- tic activities, and stability of KAIST rH IL-2 and KAIST Ser'* rH IL-2 for the establishment of pre- clinical screening system and therapeutic applica- tions 127128 Materials and Methods Production of Antiserum Against Purified Ser®*-rH IL-2 Rabbit and chick antisera against KAIST Ser!®°-rH IL-2 were obtained according to the fol- lowing immunization regimen. Each 100 ng of lyophilized protein was dissolved in 200 yl of PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.4) and mixed thoroughly with the equal volume of complete Freund's adjuvant. After emulsification, the solution was injected subcutaneously and boosted after 2 weeks of first injection. After 1 week of second boosting, formation of antibody and titers of antisera were evaluated by double immuno- diffusion analysis (Lee and Hahm, 1986) in 1% agarose Biological Activity Assay Bioassay of :H IL-2 was carried out essentially according to the standard procedure (Gillis et al, 1978) as modified by Talmadge (1985) using IL-2 dependent cell line, CTLL. The cells were cultured in RPMIL640 media containing fetal calf serum (FCS, 10%), L-glutamine (10 mM), sodium pyru. vate (1 mM), 2-mercaptoethanol (0.5 jxM), antibio- tics (1%) and 20-30% con A supernatant. Con A supernatant was prepared by stimulating mouse spleen cells with con A (20 jeg/ml) for 24 hours For assay, serially diluted IL-2 samples (100 jl) were incubated with CTLL for 24 hours, pulsed with*H-TdR (1 jCi/well), harvested (Brandel, Model M-24, MD., U.S.A.) and counted cellular radioactivity (LKB 1216 Rackbeta I Liquid Scin- tillation Counter, LKB Bromma, Sweden) after in cubation for 8 hours. The Biogen rH IL-2 (Biogen Research Corporation, Cambridge, MA., U.S.A.) was included as a reference in each assay. The Biogen rH IL-2 was shown to have an identical activity when compared to BRMP (Biological Re sponse Modifiers Program, NCI, NTH) IL-2 refer- ence reagent and the IL-2 activity for each sample was converted to reference units (RU)/ml as de- fined by the standard. The assay data were analy- zed according to the program developed by Sette et al, (1986). Augmentation of Natural Killer (NK) Cell Activity Augmentation of NK cell activity was assayed Korean Biochem. J. (1988), Vol. 21(2) Hye-Lim Choi et al. according to the standard procedure developed by the Preclinical Screening Laboratory (PSL) of Biological Response Modifiers Program (BRMP), NCI, NIH (Talmadge et al, 1985b). Spleens were removed from C3H mice and a single cell. suspen. sion was prepared via a water lysis. Cells were washed in HBSS (Hank's balanced salt solution containing calcium and magnesium), counted and the concentration was adjusted to § X 10° cells/ml in media (PRMI 1640 containing 10 mM gluta. mine, 5% FCS and 0.005% gentamycin). Cells were plated in quadruplicate in 24 well plate (Costar, U.S.A) containing rH IL-2 samples (approximately 20,000, 2,000, 200, 20 units/ml) and the plates were incubated overnight in 5% CO» at 37°C. Polyinosi- nic-polycytidylic acid and poly-L-lysine solubil- ized by carboxymethyl cellulose (Poly IC-LC, NCI) was included as a positive control. (Talmadge et al, 1985b; Talmadge and Hartman, 1985). Cells were harvested into 50 ml polypropylene tubes, washed by centrifugation and the concentration was readjusted to 5 X 10° cells/ml in fresh media be- fore plating (4 dilutions, 100 jl each, triplicate) in a 96 well U bottom microtiter plate, To each well, 100 yl of Cr°!-YAC cells (YAC cells were treated previously with chromium-51; 200 ~Ci/5 x 10° cells) was added and incubated at 37°C for 4 hours in 5% COz, the supernatant was counted in an LKB 1270 Rackgamma Il (LKB Bromma, Sweden) and the data was analyzed. Mixed Lymphocyte Reaction (MLR) Assay Allogeneic mixed lymphocyte reaction (MLR) assay was performed according to the standard pro- cedure of NCI, NIH (Talmadge ef al., 1984b), Spleens were removed from both C57 BL/6 and C3H mice and single cell suspensions were pre- pared via a water lysis. Stimulator spleen cells, C857 BL/6 cells (2 x 10° cells/ml) which were irradiated at 2,000 rad were admixed with responder spleen cells, C3H cells, (1 x 10” cells/ml) in flat-- bottomed 96 well plates. The cells were admixed in EHAA (extra high amino acid) media, supplemented with 5 x 10-5 M 2-mercaptoethanol and 0.5% nor. mal mouse serum (Click et al, 1972; Heber-Katz et al, 1972) containing different doses of rH IL-2 or thymosin fraction 5 (Hoffman-La Roche Inc., NJ. U.S.A), which served as a positive control (Tal- madge, 1984; Talmadge et al, 1984). The culturesTherapeutic Activities of rH IL-2 were pulse-labeled with tritiated thymidine(1 j«Ci/ well) for 24 hours prior to harvest at 4 or 5 days following culture initiation. The cultures were har- vested with a Brandel 24 well automatic cell harvester on to Whatman glass microfibre filters Cells were water-lysed to remove soluble pools of radiolable, and the amount of incorporated tritium- thymidine was determined using an LKB 1216 Rackbeta IT liquid scintillation counter in PCS scintillation fluid (Amersham, Buckinghamshire, U.K.). The MLR results are expressed as a relative proliferation index (RPI) (Dean et al, 1977) and stimulation index (SI) (Talmadge et al, 19842): PM(EAC)~CPM(CRC), “CPM(CAC)—CPM(CRC)* RP! CPM allogenic culture, =PM(CRC) where EAC is experimental allogenic culture, CRC is control responder culture and CAC is control allogenic culture. Evaluation of Stability of rH IL-2 KAIST rf IL-2 and KAIST Ser!?®-rH IL-2 were evaluated for their stability by analyzing pro- teins using a gradient polyacryl amide gel elec trophoresis in the presence of sodium dodecyl sul fate (SDS-PAGE) and MLR assay after storing samples for 6 months. Linear gradient (7.5-18%) SDS-PAGE was carried out using 4% stacking gel (Laemmli, 1970). Duplicate gels were run so that one gel contained samples treated with 2-mercaptoethanol and the other in unreduced states. For each lane, 15 jg of sample was loaded and proteins were visualized by staining with silver (Biorad, U.S.A.). MLR assay for the stored samples were carried out as previously described. Results and Discussion The biological activity of the purified rH IL-2 was about 1-3 x 10° units/mg protein. The activ. ity of KAIST rH IL-2 and KAIST Ser!?°-rH IL-2 was 1.0 RU x 10°/mg and 2.5 RU Xx 10°/mg pro- tein, respectively. The result (Table 1) is an aver age value obtained from various samples assayed at, least in duplicte. As the data indicates, the mutein, 129 Table 1. Activity of Natural and Recombinant Human. IL-2 IL-2 IL-2 Activity code Specific Activity Designation RU x 10°/mg protein Recombinant IL-2 KAIST 1H 1L-2 10 KAIST Ser-rH IL-2 25 R- 02 R2 LI R3 95 R4 50 Lymphoid IL-2° Na 0.02 N-2 02 Nn 765 Nea 05 *Data for the recombinant I1,~2 of R-1 through R-4 and all of the lymphoid IL-2 samples were obtained from the PSL (unpublished results) KAIST Ser!?°-rH IL-2, shows more biological activity than the wild type. Since the biological or therapeutic activity of the mutein has been reported to be comparable with that of wild type (Liang et al., 1986), the present data suggest advantages for producing mutein for preclinical and clinical stu- dies. The activity of KAIST rH IL-2 samples was ‘compared with several recombinant or lymphoid hu man IL-2 samples prepared from various laborator- ies, and the result (Table 1) indicated that our re combinant IL-2 is comparable with other products. In vitro incubation of mouse spleen cells with KAIST rH IL-2 and KAIST Ser!?-1H IL-2 at doses > 10 units/ml significantly augmented NK cell activity in vitro (Table 2). The positive control, poly IC LC, also activated NK cells in vitro, and 200 units/ml of KAIST rH IL-2 and <180 units/ ml of KAIST Ser!-rH IL-2 were required to obtain similar high levels of augmented NK cell activity. the result is superior to that obtained from Biogen rH IL-2 which showed 1,000 units/ml rH IL-2 being required to obtain similar high levels of augmented NK cell activity (Talmadge, 1985). The result also showed that the mutein, KAIST Ser'?5-rH IL-2, is better than the wild type, KAIST rH IL-2, for the augmentation of NK cell activity in. vitro The allogeneic MLR was performed at a respon. Korean Biochem. J. (1988), Vol. 21(2)130 Table 2. KAIST rll IL-2 L and KAIST Ser'®°-rH IL-2 augmentation of Hye-Lim Choi et ab Percent cytotoxicity (YAC), Agent Concentration Lytic 50:1 25:1 121 61> Units Medium - 12,0 0 0 PolyIC-LC 1 ug/ml 519° 299° 190" 74 7 10 pg/ml 59.3 475° 24.2" 111° 10 KAIST rH IL-2 2 U/ml 372 225" 118" 66" 4 20 U/ml 46.7 433" 242 128 8 200 U/ml 45.1 50.4" 35.2 187" 9 2 18 U/ml 37.7" 216° 130" 49 4 180 U/mi 60.6" 439° 293° 166 4 1,800 U/ml 71.9 59.4" 455° 268" 17 “Significant increase in cytotoxicity; “Indicates effector to target ratio. P < 001 (paired Student's t test) Table 3. KAIST rH IL-2 and KAIST Ser'#-rii IL-2 augmentation of lymphocyte a mixed lympho ‘te response (MLR)* Medium Agent Dose C3H only 1,084 Thymosin FS 50 eg/ml 11,983 KAIST rif IL-2 1,800U/ml 25,734 KAIST Ser!?°-rH IL-2 200U/ml 44,407 C3H_ Stimulation Relative +CS7BL/6 index index 12.999 1.00 1.00 173% 137" 1.36 39.847 23.56" 325° 47903 42.12 392° "Medium control was in the absence of any was thymosin fraction 5 (F5). "The values are expressed in counts per minute (cpm) and averaged from quadruplicate samples. paired Student's test der/stimulator ratio of 5:1 to demonstrate maximum immunomodulation (Talmadge et al, 1984a). When responder and stimulator spleen cells were cocul- tured in the presence of KAIST recombinant IL-2 samples or the postive control thymosin fraction 5 (Table 3), they significantly increased the incor poration of tritium-thymidine into the allogeneic mixed lymphocyte cultures. Maximal stimulation was observed at 1,800 units/ml for KAIST rH IL-2 and 200 units/ml for KAIST Ser!°-rH IL-2, with a stimulation of the MLR greater than that observed with the positive control, Since the dose tested was not minimum and the minimum dose to give a significant increase in the allogeneic MLR was not determined, additional experiments using lower doses of IL-2 samples are currently being carried out. KAIST rH IL-2 and KAIST Ser!*5-rH Korean Biochem. J. (1988), Vol. 21(2) immunomodulator, and the positive control *p<001 compared with the control group by IL-2 were a 0 found to be blastogenic when incu: bated with C3H spleen cells alone (jie., in the abs- ence of mitogen or allogeneic stimulation). The re- sult is comparable to the results obtained with other rH IL-2 (Talmadge, 1985; Thurman et al, in press) The result also indicated that the mutein, KAIST Ser'*-rH IL-2 is more active than the wild type, KAIST rH IL-2, in the augmentation of MLR which is consistent with the other results currently obtained, implying the advantages for producing mutein. Although both rabbit and chick were immunized with KAIST Ser!*-rH IL-2, chick only produced antibody as determined by double immunodiffusion analysis (Fig. 1). It was also observed that the re- combinant protein when reconstituted with water was only reactive and formed precipitin lines withTherapeutic Activities of rH IL-2 Fig. 1. Double immunodiffusion of rH IL-2 against chick anti-rH IL-2 serum. Antiserum was two-fold serially diluted from A to F. chick antiserum in acidic pH. When higher pH than 5.5 was used, no precipitation reaction with anti body was observed indicating that the IL-2 mole- cule is stable and maintains its natural conforma. tion at acidic pH and at neutral or alkaline pli it aggregates. 66 K- 45 K- ee 20 K- 14.2K — 1234 5678 131 ‘The stability of the KAIST rH IL-2 and KAIST Ser'®rH IL-2 was also tested by SDS-PAGE Gradient gel electrophoresis was carried out in order to observe any by-products formed in wide molecular weight range. Although silver stain is generally known to require less than 100 ng of pro- tein to be visualized, more than 10 yg of each sample was loaded in order to be able to detect any minute quantity of either degradation or aggrega- tion products which might be formed during the storage. The result (Fig. 2) showed single protein band of IL-2 for each sample which means that there is no degradation or aggregation product formed whether the samples were treated with 2-mercaptoethanol or not indicating that both KAIST rH IL-2 and KAIST Ser!°-1H IL-2 are very stable The stability in terms of therapeutic activity was assessed by performing MLR assay using identical samples before and after storage for 6 months. The result (Fig. 3) showed that although there was some loss of biological activity, both KAIST recom: binant proteins were able to maintain significant therapeutic activities. Therefore, the present study shows that both KAIST rH IL-2 and KAIST Ser'°-rH IL-2 are suitable for preclinical and clinical studies. Using KAIST Ser!5-rH IL-2, phase I and II clinical stu- 1234 5678 Fig. 2. Gradient SDS-PAGE (7.5-18%) of rH IL-2 and Ser'®®-rH IL-2. Panel A shows the result obtained from the samples treated with 2-mercaptoethanol and panel B shows the result obtained from unreduced samples. Lane 1-4 shows rH IL-2 puri fied under reducing (lanes 1 and 2) and nonreducing (lanes 3 and 4) conditions and lane 5-8 shows Ser!®-rH IL-2 purified under reducing (lanes-5 and 6) and nonreducing (lanes 7 and 8) conditioas. Korean Biochem. J. (1988), Vol. 21(2)182 MLR O ser 60 @ 10/28/87 8 15 10 stimulation index ($1) relative proliferation index (RP1)* SSS SSSA AAA y i / / Yj y / / / ] IS SSS thymosin Ser!25 wild gen FH- IL-2 (109u/mi) Fig. 3. Augmentation of lymphocyte response in mixed lymphocyte reaction (MLR). Medium control was in the absence of any immunomodulator, and the positive control was thymosine fraction 5(F5) Ser!°rH IL-2, rll IL-2, and rH IL-2 from Biogen co, are abbreviated as Ser!?°, wild and Biogen. re- latively. dies are being carried out at various medical in: stitutions. References Click, R-E., Benck, L., and Alter, B.J. (1972) Cell. Im- munol. 3, 264 Korean Biochem. J. (1988), Vol. 21(2) Hye-Lim Choi et al, Dean, J.H., Connors, R., Herberman, R.B., Silva, J., Old ham, R.K., and McCoy, J.L. (1977) Int. J. Cancer 20, 359. Gillis, S., Far, M-M., Ou, W., and Smith, K.A. (1978) J. Immunol. 120, 2027. Han, M.H., (1987) in annual Report submitted to the Ministry of Science and Technology, KOREA Heber-Katz, E., and Click, R.E, (1972) Cell. Immunol. 8, 410. Laemmli, U.K, (1970) Nature (London) 227, 680. Lee, M.K., and Hahm, K-S. (1986) Korean Biochem. J. 19, 399. Liang, S.M., Allet, B., Rose, K., Hirschi, M., Liang. C.M,, and Thatcher, D.R, (1985) Biochem. J. 229, 429, Liang, S.M,, thatcher, D.R., Liang, C.M,, and Allet, B. (1986) J. Biol. Chem. 261, 334 Mihich, E., and Fefer, A. (1983) in biological Response Modifiers sudcommittee report. In US. Government Printing Office (eds.); “National Cancer Institute Monograph 63” Washington D. C. Oldham, P.K., and Smally R. V. (1983) J. Biol. Resp. Mod- #21 Sette, A., Adorini, L., Marubini, E., and Doria, G. (1986) J. Immunol. Methods 86, 265. Talmadge, J. E. (1984) in Chemical Regulation of Immun. ity in Veterinary Medicine, pp 457-465, Alan R. Liss, Inc.. New York. Talmadge, J.E. (1985) J. Biol Resp. Modif. 4, 18. Talmadge, J.E.. Adams, J., Philips, H., Collins, M., Lenz B. Schneider, M., and Chirogos, M. (1985a) Cencer Res, 45, 1066 Talmadge. J.E., Benedcit, K.L., Uithoven, K.A., and Lenz, B.E. (1984a) Immunopharmacol. 7, 17. Talmadge, J E., Fidler, IJ. and Oldham, R. K. (1985b) ia Screening for biological response modifiers. Methods and Rationale, Boston. Martinus Nihjoft Talmadge. J. E., and Hartman, D. (1985) J. Biol. Res. Mod- if 4, 484. Talmadge. J.E.. Uithoven, K.A.. Lenz, B.F., and Chie gos. M., (1984b) Cancer Immunol. Immunother. 18, 185, Terry, W.D., and Rosenberg S. A. (1982) in Immunother: apy of Human Cancer, New York, Excerpta MedicaTherapeutic Activities of rH IL-2 133 ZR ola CIELF2ZI-2: I. WS WAIN IS Aaa» Salg « James E. Talmadge’ - B44 (UI HIEU HAS Ge Dat Al, ‘Preclinical Screening Laboratory, NCI-FCRF, Frederick MD., U.S.A.) REL Bey Belaalal yee lal abel Pzl-2 (KAIST ell IL-2, KAIST Ser'?*-rHl IL-2) AUAAG FY also] S8e7] HE SASS AeA Y aad dgse Sasi. In vitro 4 abel fol] AEBS] SALSA YLT VITUS Aol dS ehwtoot cE saps} AAG VABL-2 ASE) HAE ats Ach AST al Veh F229] Vaal yesh AVES VAS 248b1 satel dala AA al UH Fal-2o] AE YQIS Fa IL 2m] SAAS ABA MSG al el Fal-29} 6] Qa se} WSS Ssh gBonl, Kak oY ADA Led 21-22] VIAL EY UHPWS Qavids wes 2 a4 yaad age SAVE ETUIT USA fala] WAS vel Falocl, arigga Valsad Saal sol AAA] BELL Melsigteh, of 49] Boke KAIST aaa ala) Ve Fal-27t AYAAY Bhd @ Sol Ssh lol AWS 44H Korean Biochem. J. (1988), Vol. 21(2)