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Human Interleukin 2 Analogues That Preferentially Bind the Intermediate-Affinity Interleukin 2 Receptor Lead to Reduced Secondar

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Human Interleukin 2 Analogues That Preferentially Bind the Intermediate-Affinity Interleukin 2 Receptor Lead to Reduced Secondar [CANCER RESEARCH 53. 2597-2«)2. June I. 1993] Human Interleukin 2 Analogues That Preferentially Bind the Intermediate-Affinity Interleukin 2 Receptor Lead to Reduced Secondary Cytokine Secretion: Implications for the Use of These Interleukin 2 Analogues in Cancer Immunotherapy ' Keith M. Heaton,2 Grace Ju, and Elizabeth A. Grimm neiHirnneiiis ctf Tumor rìitìlogydittiGeneral Surgen; the University of Te\as M. D. Anderson Ccincer Center. Hmtuon. Te\(ts 77030 fK. M. H.. E. A. G.J. anil the Department of Inflamtiiiition/Aittotinintine Diseii\e.\. HvffnHinn-lMRfciie. Inc.. M/f/cv. New Jersey 07110 ¡G.J.¡ ABSTRACT Many of the signs and symptoms of IL-2-induced toxicity are thought to be caused by the actions of cytokines such as IL-lß. Cancer patients undergoing interleukin (IL)-2-based immunotherapy TNF-a, TNF-ß.and IFN-y released by IL-2-activated PBMC (9-14). frequently experience dose-limiting side effects believed to be caused by the actions of such cytokines as II.-I/ÃŽ,tumor necrosis factor (TNF)-a and If secretion of these secondary cytokines could be reduced, patients -ß,and interferon-y (IFN-y). Human peripheral blood mononuclear cells receiving IL-2 immunotherapy might be able to tolerate higher and (PBMC) or monocyte-dcpleted peripheral blood lymphocytes »erestim more effective doses of IL-2. ulated for up to 7 days by either of 2 IL-2 analogues (R38A or F42K) that R38A and F42K. 2 human IL-2 analogues that have altered IL-2Ra bind to the intermediate-affinity II.-2/jy receptor but have reduced abil binding domains, differ from human rIL-2 by a single amino acid ities to bind the high-affinity II -2 receptor. We previously reported that substitution. In this report, we examined the capacity of R38A and these 11-2 analogues retain the ability to generate lymphokine-activated F42K to induce the secretion of IL-lß.TNF-a, TNF-ß,and IFN--y. killing by PBMC. In this study, we analyzed the cytokine content of Compared with cultures stimulated by rIL-2, cultures exposed to supernatants from stimulated PBMC and peripheral blood lymphocyte either of the IL-2 analogues showed significant reductions in the cultures by enzyme-linked immunosorbent assay. The secretions of IL-Iß, TNF-a, and -ß,and IFN-y induced by either R38A or F42K were mark amounts of these cytokines present. Previously, we showed that ana edly reduced compared with secretions produced in response to recombi logue-stimulated PBMC retain substantial LAK activity (15) (Table nant wild-type IL-2. In 4 experiments, secretion was reduced an average 1). Thus, the use of IL-2 analogues specific for the intermediate- of 39% for IL-lß, 57% for TNF-a, 83% for TNF-ß,and 86% for IFN-y. affinity IL-2R may prove to be an effective and less toxic means of Polymerase chain reaction analysis of recombinant wild-type 11-2 or an cancer immunotherapy. alogue-stimulated PBMC did not reveal the presence of IL-2 mRNA; thus, differential production of endogenous IL-2 could not account for these Findings. These data suggest the interaction of IL-2 and the high-affinity MATERIALS AND METHODS IL-2 receptor on human PBMC or peripheral blood lymphocyte is re quired for maximal secretion of IL-lß, TNF-a, TNF-ß,and IFN-y. Be PBMC Preparation. PBMC were obtained from normal, healthy volun cause such cytokines are believed to mediate the toxicity seen with 11-2- teers hy leukopheresis. The contents of the pheresis hags were diluted with based immunothérapies, IL-2 analogues with reduced binding to the high Hanks' balanced salt solution (Ha/relton Biologicals, Inc.. Lenexa. KS) and affinity IL-2 receptor may prove to be an effective and less toxic means of fractionated on Histopaque (Sigma Chemical Co.. St. Louis. MO) by centrif- cancer treatment. ugation at 450 x g for 30 min. PBMC were isolated at the interface, removed carefully, and washed 5 times in Hanks' balanced salt solution before being resuspended in serum-free AIM-V medium (Gibco Laboratories. Grand Island. INTRODUCTION NY) containing 2 ITISIi-glutamine. For experiments in which FBI, were used. The potent oncolytic properties of IL-21 as a single agent and in PBMC were either subjected to plastic adherence for 2 h or passed through a combination with IL-2-stimulated LAK cells have been used in many nylon wool column. PB1. were analy/ed for contaminating mnnncyte content cancer immunotherapy protocols. In a number of cancer centers, the by fluorescence-activated cell sorter using anti-LeuM3 antibodies. The per overall response rates for these IL-2-based regimens have been be centage of residual macrophages in each sample was less than 0.4%. tween 15 and 30% with complete responses in approximately 5-10% IL-2 and IL-2 Analogues. Clinical-grade human wild-type rIL-2 was ob of patients (\-4). The problem with these protocols, however, has tained from Dr. F. Khan of the Bioprocess Department at Hoffmann-LaRoche, Inc. (Nutley. NJ). IL-2 analogue proteins were prepared by site-directed mu- been the high incidence of multisystem toxicity that occurs in an IL-2 dose-dependent manner. Patients treated with IL-2-based therapies tagenesis. expressed in F.scherichia culi, and purified as described by gel filtration (16). This gel filtration technique also extracts any endotoxin that is may experience such difficulties as hypotension, cardiac arrhythmias, present in the purified protein preparations. The IL-2 analogue protein R38A pulmonary insufficiency, and renal failure with treatment-related mor contains an alanine substitution for Arg38. and the analogue F42K has a tality rates of about 2% (I. 5-8). substitution of lysine tor Phe42 (17). Binding of the analogues to the human IL-2R was evaluated as described hy Sauve et al. (17). As previously deter mined in murine cytotoxic T-lymphocyte line proliferation assays, the specific Received 1/18/93; accepted 3/22/93. The costs (if publication ol' this article were defrayed in part hy the payment of page activities of the analogue proteins were 2 x IO7 units/mg rIL-2. 7.ft x IO6 units/mg R38A. and 3.2 x IO5 units/mg F42K (17). Because of these differ charges. This article must therefore he herehy marked advertisement in accordance with 18 U.S.C. Section 1734 solely lo indicate this fact. ences in specific activity, equimolar concentrations of each protein were used 1Supported hy NIH Grant CA45445 (E. A. G.). National Cancer Institute General in all experiments. Dose titration curves for the analogues' induction of LAK Surgery Training Grant 5 T32 CA095(XM)4 (K. M. H.I. and Core Grant CAI6672. 2 To whom requests for reprints should he addressed, at the Department of Tumor activity were presented previously (15). In those studies, maximal activity was Biology. Box 79. The University of Texas M. D. Anderson Cancer Center. 1515 Holcomhe found at the concentration of I mi: therefore. I mi concentrations were used in Boulevard. Houston. TX 77030. all further experiments. Conversion factors for rIL-2 and the analogues are: I 'The abbreviations used are: IL. interleukin: LAK. lymphokine-activated killer; mi = 308 units/ml rIL-2 =117 units/ml R38A = 5 units/ml F42K. PBMC. peripheral blood mononuclear cells; TNF. tumor necrosis factor: IFN. interferon; Activation of LAK Cells. PBMC or PBL were cultured at IO6cells/ml in rlL-2. recombmani wild-type human interleukin 2: IL-2R. interleukin 2 receptor: PBL. peripheral blood lymphocytes; ELISA, enzyme-linked immunosorbent assay; PCR. poly- AIM-V medium and incubated at 37°Cin a humidified 5% CO? atmosphere merase chain reaction. with I nvi rIL-2 or IL-2 analogue. The AIM-V medium was selected because 2597 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1993 American Association for Cancer Research. IL-2 ANALOGUE-INDUCED CYTOK1NE CASCADE it is chemically defined and serum-free, enabling us to avoid serum-induced days, the cell-free culture supematants were tested for cytokine con secondary cytokine production from the stimulated cells. centration using ELISA methodology. In 2 of 4 experiments per Cytokine Assays. Supernatant cytokine concentrations were determined by formed using rIL-2 or analogue-activated PBMC from different do ELISA detecting TNF-a and TNF-ß(R & D Systems. Inc.. Minneapolis. MN). nors, no detectable IL-Ißwas found in the culture supematants. In the IL-lß(AMAC. Inc.. Westbrook. ME), and IFN--y(Endogen, Inc.. Boston. MA) 2 experiments in which IL-Ißwas present, however, the amount of following all manufacturer protocols. Briefly, supematants were obtained from IL-Ißsecretion induced by R38A averaged only 76%. and F42K- stimulated PBMC or PBL on the indicated day and cemrifuged at 90 x g for induced IL-Ißwas about 61% of that produced in response to rIL-2 5 min to remove any cells. These supematants were incubated in duplicate or in triplicale in antibody-coated wells of the supplied microtiter plates. A second (Fig. \A). Fig. Ißshows the kinetics of IL-Ißproduction for one distinct cytokine-specific antibody/en/yme conjugate was then added to each representative experiment. IL-Ißproduction peaked at 24 h and then well followed by addition of the appropriate chromogenic substance. Absor- subsequently decreased. Supematants from the monocyte-depleted bance was determined by spectrophotometry. and the concentration of cytokine PBL cultures were also tested for IL-Iß.No detectable IL-Ißwas was determined from a standard curve. Mean cytokine secretion was deter present up to 7 days after rIL-2 or analogue stimulation in the 2 mined by standardising the analogue-stimulated cytokine secretion as a per experiments performed.
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