A simple method for the evaluation of receptor binding capacity of modified cytokines

A simple method for the evaluation of receptor binding capacity of modified cytokines

Journal of Immunological Methods, 166 (1993) 177-182 © 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00 177 JIM06852 A...

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Journal of Immunological Methods, 166 (1993) 177-182 © 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

177

JIM06852

A simple method for the evaluation of receptor binding capacity of modified cytokines M a r c o Chianelli a, Alberto Signore a, R a y Hicks c, R o b e r t o Testi b, Marcello Negri a, P e t e r C.L. Beverley ,,c " Clinica Medica II, University 'La Sapienza', and b Dipartimento Medicina Sperimentale e Scienze Biochimiche, University 'Tor Vergata', Rome, Italy, and c Imperial Cancer Research Fund Tumour Immunology Unit, University College London Medical School, 91 Riding House Street, London W1P 8BT, UK

(Received 7 April 1993, revised received 21 June 1993, accepted 8 July 1993)

We have developed a flow cytometric method to evaluate the binding of interleukin-2 analogues to receptors. The method relies on competition for binding between a fluorescein-conjugated monoclonal antibody (MoAb) directed against the human interleukin-2 receptor a chain (fluorescein isothiocyanate (FITC) anti-IL-2R) and the test protein. IL-2R positive ceils are incubated with FITC-anti-IL-2R MoAb in the presence of native IL-2 or IL-2 iodinated by either the chloramine-T or the lactoperoxidase-glucoseoxidase method. The binding of IL-2 is indicated by decreased fluorescence. This method is suitable for measuring the binding capacity of modified IL-2 molecules and avoids the need for radioactive tracers. It provides a simple and reproducible technique, which can be extended readily to the study of the receptor binding capacity of cytokines conjugated with toxins, drugs or other molecules. Key words: IL-2; Cytokine; Fluorescence activated cell sorter; Iodination; IL-2R

Introduction

Cytokines are used in both basic and clinical sciences. Radiolabelled cytokines are important tools for the detection of specific cytokine receptors on defined cell populations and have been used recently for in vivo diagnostic purposes

* Corresponding author. Abbreviations: a-LA, a-lactalbumin; BSA, bovine serum albumin; CT, chloramine-T; FITC, fluorescein isothiocyanate; IL-2, interleukin-2; IL-2R, interleukin-2 receptor; LPO/GOD, lactoperoxidase/glucoseoxidase; MoAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; PMA, phorbol myristate acetate; PHA, phytohaemagglutinin; PBS, phosphate buffered saline.

(Robb et al., 1985; Signore et al., 1987, 1992; Chianelli et al., 1993). Moreover, in certain cancers, cytokines are used as immunotherapeutic drugs, alone or conjugated with toxins (Borden and Sondel, 1990; Lorberboum-Galski et al., 1990). An important requirement for effective use of cytokines is the retention of binding capacity for their specific receptors. During radiolabelling reactions or conjugation with toxins, the molecular structure of cytokines can be damaged and impaired binding may result. So far cytokine binding to specific receptors has been evaluated using radiolabelled analogues and Scatchard analysis of data (Robb et al., 1985). This method provides useful information on receptor number and affinity but a major limitation

178 is that results obtained using radiolabelled cytokines may be affected by impaired binding of the radioactive cytokine, due to the radioiodination procedure. The aim of this study was to set up a simple method to evaluate the receptor binding capacity of modified cytokines by comparison with the native molecule. The method does not require the use of radioactive substances and any modified cytokine may be easily studied. The technique is based on competition of the test cytokine with a fluorescein conjugated monoclonal antibody directed against the cytokine receptor for binding to cells expressing the receptor for the test cytokine. Since cell bound fluorescence, measured by flow cytometry, is proportional to the binding of the fluorescein isothiocyanate (FITC) MoAb, a decreased fuorescence intensity in the presence of the cytokine is interpreted as binding to its receptor. The receptor binding capacity of a modified cytokine is then compared with the binding capacity of the native cytokine. We have applied this technique to interleukin-2 (IL-2) and have studied the effect of different iodination techniques on the capacity of IL-2 to bind to its receptor.

Materials and methods

Purification and culture of interleukin-2 receptor positive cells IL-2 receptor (IL-2R) positive cells were generated from peripheral blood mononuclear cells (PBMCs) isolated from healthy donors by centrifugation over Ficoll-Hypaque. Cells were cultured for 48 h at 106/ml in complete culture medium (RPMI 1640 with L-glutamine 30 mg/100 ml, fetal calf serum (FCS) 10%, and streptomycin and penicillin, 100 U / m l ) with 1 / z g / m l purified phytohaemagglutinin (PHA) (Wellcome) at 37°C. Prior to use, cells were washed twice and resuspended in cold phosphate buffered saline (4°C) with 1% bovine serum albumin (PBS-BSA). As a positive control an IL-2R expressing human hybridoma (IA3-4) was used (Ando et al., 1985). Before testing, cells at 5 × 105/ml were incubated at 37°C, for 30 min with PMA (3

ng/ml), washed three times and cultured in complete medium for 24 h at 37°C. Before use they were washed twice and resuspended in cold PBSBSA.

Fluorescent anti-IL-2R monoclonal antibody Expression of IL-2R was determined by flow cytometry using a mouse IgG1 FITC anti-IL-2R chain MoAb (2A3, Becton Dickinson). This MoAb recognises the IL-2 binding site on the a chain of human IL-2R (Urdal et al., 1984). Thus, IL-2 and 2A3 are cross-inhibiting molecules and the MoAb was titrated in order to determine the minimum concentration giving saturation of receptors. Decreasing concentrations (2.08-0.001 ~ g / m l ) of the FITC anti-IL-2R MoAb were incubated for 30 min at 4°C with IL-2R positive PBMCs. After washing, cells were resuspended in 200 /zl of cold PBS-BSA and analysed on a flow cytometer (FACScan, Becton Dickinson). For evaluation of background non specific fluorescence a control mouse IgG1 FITC MoAb directed against an irrelevant antigen was used.

Inhibition of receptor binding of FITC anti-IL-2R by IL-2 The binding capacity of human recombinant IL-2 (Glaxo, UK and EuroCetus, Netherlands) was calculated by its ability to inhibit the binding of FITC anti-IL-2R MoAb to activated cells. IL-2 (40/~l) was incubated at different concentrations (1-10,000 nM) with 10 /~l of PHA activated PBMCs or PMA activated IA3-4 (105 cells) at 4°C. After 30 min 10 /.d of FITC anti-IL-2R MoAb (0.3 /zg/ml) were added for a further 30 min. Cells were washed twice, resuspended in 200 /~1 of cold PBS-BSA and analysed by flow cytometry. As a positive control FITC anti-IL-2R MoAb (0.3/~g/ml) was incubated in the absence of IL-2 as described above. As a control for the specificity of inhibition, FITC anti-IL-2R MoAb was incubated with different concentrations (1200, 300, 75 nM) of a-lactalbumin (cr-LA), an irrelevant protein of the same molecular weight as IL-2. As a further control, IL-2 (1200, 300, 75 nM) was incubated with a FITC anti-CD3 MoAb

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(anti-Leu 4, Becton Dickinson) at a concentration of 3 ~ g / m l . FITC anti-CD3 MoAb had been previously titrated (25-0.2 /zg/ml) to determine the concentration giving saturation of CD3 binding sites, as described for FITC anti-IL-2R MoAb.

Iodination of IL-2 and assay of its binding capacity The binding capacity of IL-2 was tested after iodination with non-radioactive iodine-127 (1271) in order to evaluate the effect of different iodination techniques. IL-2 was labelled using the following methods: lactoperoxidase (LPO) in combination with glucose oxidase (GOD) and chloramine-T (CT). LPO method. IL-2 (5 /zg) was incubated for 15 min with LPO (0.5 /zg), GOD (0.05/~g), NaI (0.6 nmol) and glucose (50/xg). Chloramine-T method. IL-2 (5 /zg) was incubated with NaI (0.6 nmol) and chloramine-T (10 /xg); after 20 s sodium metabisulphite (40/zg) was added to stop the reaction. The reaction mixture was then diluted to 179/~1 in cold PBS-BSA and IL-2 used for binding assay. As a control for the effectivness of the iodination, in separate experiments, IL-2 was labelled with radioactive 12~I (Amersham) using the methods described above, and the labelling efficiency calculated. The binding capacity of iodinated IL-2 was tested in parallel with unlabelled IL-2 by its ability to inhibit the binding of FITC anti-IL-2R MoAb as described above. Flow cytometric analysis Cell bound fluorescence was measured with a flow cytometer (FACScan, Becton Dickinson). Fluorescence data were collected using a logarithmic scale after gating on forward and right angle light scatter, in order to discriminate between viable and non-viable cells. Binding of FITC-MoAbs was displayed as fluorescence profiles (5000 ceils analysed per histogram) and quantified as mean fluorescence channel number. Mean channel number was transformed to give linear values in the range 1-10,000, using the formula: linear fluorescence value = 10 [(c/1°23) x 4]

where C is the mean channel number as produced by FACScan software. After linearisation as above, specific binding was calculated by subtraction of the background value obtained with the non-specific antibody from that obtained with FITC anti-IL-2R.

Results

After 48 h stimulation with PHA, 75% of PBMCs expressed IL-2R as determined by FITC anti-IL-2R MoAb. More than 50% of PMA activated IA3-4 cells expressed IL-2R. Staining of IL-2R positive cells with FITC anti-IL-2R MoAb was maximal at concentrations from 2.48 to 0.3 /xg/ml. With lower MoAb concentrations fluorescence decreases progressively (Fig. 1) and 0.3 /zg/ml was therefore used in further experiments. The FITC anti-CD3 control MoAb was used at 3 p.g/ml, the lowest concentration giving maximal binding. Incubation of activated PBMCs with increasing concentrations of IL-2 followed by the addition of the FITC anti-IL-2R MoAb showed a progressive decrease of fluorescence intensity. Fig. 2 shows a typical set of fluorescence profiles from such an experiment, in which the scatter gate for analysis included both small lymphocytes (largely unstained) and T cell blasts, which stain brightly with the FITC anti-IL-2R MoAb. Fig. 3 shows the dose dependent inhibition of the binding of the MoAb by a different (and more potent) batch

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of IL-2. The same dose dependent inhibition of F I T C anti-IL-2R M o A b binding was observed when IL-2 was incubated with P M A stimulated IA3-4 cells. Very little inhibition of F I T C anti-IL-2R MoAb was observed at IL-2 concentrations less than 50 nM. A range of concentrations between 75 and 1000 nM was therefore used for comparison of the binding capacity of unlabelled and iodinated IL-2. Control experiments using a - L A with F I T C anti-IL-2R M o A b or IL-2 with anti-CD3 MoAb did not show any decrease of fluorescence intensity with respect to the positive controls. Incubation of cells with IL-2 iodinated using the L P O / G O D method followed by the addition of F I T C anti-IL-2R M o A b caused a dose dependent decrease of fluorescence intensity comparable with that induced by native IL-2 (70 vs. 71% decrease at 1200 nM IL-2). IL-2 iodinated using the chloramine-T method was poorly inhibitory (18 vs. 71% decrease at 1200 nM IL-2) (Fig. 4). This difference was not due to a difference in iodine incorporation since the labelling efficiency obtained with the two methods was comparable, 75% using the CT method and 80% using the L P O / G O D method).

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Discussion It is important to evaluate the effect of iodination or other procedures on the binding capacity of cytokines and to determine the best method for production of the most active receptor binding modified cytokine. Moreover it is necessary, for a proper interpretation of results, to calculate the binding per molecule of modified compared to unmodified cytokine. The titration curve of FITC anti-IL-2R showed that 0.3 /zg/ml is the lowest concentration that gives near-maximal fluorescence intensity. The establishment of this concentration is critical in order to obtain maximal sensitivity of the method. While others have used fluorescent cytokines to detect receptor binding and shown that this can be inhibited by unlabelled material (Sonberg et al., 1992), the use of such a method for evaluation of modified cytokines has the inherent disadvantage that the fluorescent cytokine is itself modified. It would, of course, be possible to compare the ability of modified and unmodified cytokines to inhibit binding of the fluorescent cytokine but the present method has the advantage of using relatively cheap and stable fluorescent MoAb instead. However the properties of appropriate MoAbs deserve some comment. Most MoAbs have binding constants in the nM to /xM range, overlapping with the affinities of cytokines (nM-pM range). Thus at concentrations of MoAb at, or just below saturation, displacement of MoAb does not require an excessive concentration of cytokine. Clearly the MoAb must also bind to an appropriate epitope of the cytokine receptor. The 2A3 MoAb used in these experiments is known to bind to the IL-2R a-chain, which is a part of both high and low affinity IL-2R, and so may interfere with binding to both types of receptor. Although no precise affinity is available for 2A3, an initial titration to determine the saturating concentration for binding obviates the need to know this. This is borne out by the observation that very similar inhibition data was obtained using the anti-Tac IL-2R MoAb (unpublished data). Our data indicate that the technique permits the evaluation of the binding capacity of IL-2 to specific receptors and can distinguish the recep-

tor binding capacity of different analogues of IL-2. The effect of physical and chemical agents on cytokines can therefore be evaluated without the use of radioactive analogues. In the present study we have shown that IL-2 iodinated by the L P O / G O D method fully retained its receptor binding capacity while material labelled by the chloramine-T procedure exhibited poor activity. This technique is simple and reproducible. It requires only the availability of a MoAb directed against the cytokine binding site of a cytokine receptor and the relevant cytokine in micromolar quantities. The method should be easily applicable to other modified cytokines, several of which have already been used in vivo (Borden et al., 1990; Lorberboum-Galski et al., 1990).

Acknowledgements M.C. is the recipient of a fellowship from the Pasteur Institute 'Fondazione Cenci Bolognetti'. This work was partially supported by C.N.R. research grant no. 920357.04. We are grateful to Dr. E. Zanders (Glaxo, UK) and Dr. F. La Monica (EuroCetus, Netherlands) for their kind gifts of recombinant IL-2.

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182 Signore, A., Beverley, P.C.L., Parman, A., Negri, M. and Pozzilli, P. (1987) Labelling of interleukin-2 (IL-2) with 123-iodine with retention of its capacity to bind to activated lymphocytes. Exp. Clin. Endocrinol. 89, 301. Signore, A., Chianelli, M., Toscano, A., Monetini, L., Tonnarini, G.F., Nimmon, C.C., Britton K.E., Pozzilli, P. and Negri, M. (1992) A radiopharmaceutical for imaging areas of lymphocytic infiltration: 123I-interleukin-2: labelling procedure and animal studies. Nucl. Med, Commun. 13, 713.

Sonberg, R.L., Robinson, J.P. and Felssburg, P.J. (1992) Detection of canine interleukin-2 receptors by flow cytometry. Vet. Immunol. Immunopathol. 33, 17. Urdal, D.C., March, C.J., Gillis, S., Larsen, A. and Dower, S.K. (1984) Purification and chemical characterisation of the receptor for interleukin-2 from activated human T lymphocytes and from a human T cell lymphoma cell line. Proc. Natl. Acad. Sci. USA 81, 6481.