IDENTIFICATION OF STRUCTURAL DIFFERENCES BETWEEN DIFFERENT FORMS OF INTERLEUKIN-2 (IL-2) USING ANTI-(HUMAN RECOMBINANT) IL-2 MONOCLONAL ANTIBODIES Marcel L.C.M. Mevissen,‘,* Mark de Boer,2S3Kees Tuin,’ Joseph M. Tager,2 Cornelis de Groot’ We have generated a panel of murine monoclonal antibodies (mAbs) directed against recombinant human interleukin-2 (rh IL-2). All mAbs have similar affinities (YE = lo-* M) and are of the IgG, isotype. Specificity of the mAbs has been established using an ELISA method and immunoprecipitation of native human interleukin-2 (nh K-2) present in supernatants from PHA-stimulated human mononuclear cells (PBMNC). Four of the nine mAbs inhibit the rh IG2-dependent proliferation of a murine cytotoxic T-lymphocyte line (CTLL-2). The estimated ID,,, in the presence of 0.75 IU rh IL-2/ml, ranges from 2.0 p&ml to 30 &ml final concentrations of the antibodies. Using different forms of IL-2 we found that the mAbs give different patterns of inhibition of the IL+dependent proliferation. One mAb (AMCIB 27) is able to discriminate between rh and nh IL-2. Findings with another mAb (AMCIB 24) indicate that possible functional differences between human IL-2 and recombinant murine (rm) IL-2 are caused by differences in the active sites. The results of this investigation show that it is possible to obtain mAbs, after immunization with rh IL-2, that differ in their ability to inhibit the biological action of different forms of IL-2. Copyright o 1991 by W.B. Saunders Company
Interleukin 2 (IL-2) is a lymphokine produced by activated human T lymphocytes.‘.’ IL-2 promotes the proliferation of activated T cells, including cytotoxic T cells.3l4In addition, IL-2 promotes the proliferation of other lymphocytes such as natural killer cells,5~6activated B cells,‘x8and lymphokine-activated killer cells.9Y10 IL-2 mediates its growth-promoting effects by interaction with a high-affinity receptor. This high-affinity receptor is composed of at least two different glycoproteins: a low-affinity receptor (Tat-antigen, ~55) and the p7O/p75 antigen.“.l4 In addition, there are indications that the receptor could have a more complex structure. Several authors have identified a number of glycoproteins that are closely associated with the IL-2 receptor.‘5.‘6 The functional role of these glycoproteins remains to be elucidated.
The production of three useful monoclonal antibodies (mAbs) directed against purified native human IL-2 has recently been achieved.” Recombinant DNA technology has made it possible to obtain large amounts of active recombinant human (rh) IL-2. Due to glycosylation, the molecular weight of native human (nh) IL-2 is about 1 kD higher than that of the recombinant form.18 So far, no functional differences are known between recombinant and native human IL-2. In the present study we are the first to report on a group of mAbs raised against rh IL-2. These mAbs recognize both native human IL-2 and rh IL-2 and differ in their ability to inhibit the biological action of the different forms of IL-2.
RESULTS Hybridoma Mono&ma1
‘Laboratory of Cell Biology and Histology and *EC. Slater Institute for Bibchemical R&earch, Acadkmical Medical Center, Meibergdreef 15. 1105 AZ Amsterdam, The Netherlands. ‘Present adhress: Department of Immunology, Cetus Corporation, Emeryville, California, USA. *To whom correspondence should be sent. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0301-0005$05.00/O KEY
54
WORDS:
IL-Zimonoclonal
antibodies/structural
differences
and Ascites Fluid Production for Antibodies with Specificity for rh IL-2
Ten days after the second booster, serial dilutions of the serum of one mouse were tested for inhibition of the action of IL-2. We found that increasing concentrations of serum decreased the rh IL-2-dependent proliferation of the CTLL-2 cells (data not shown). Two weeks after fusion, the culture supernatants were tested for specific total immunoglobulin production. CYTOKINE,
Vol. 3, No. 1 (Januaxy),
1991: pp 54-59
Monoclonal antibodies raised against rh IL-2 / 55
Table 1. Characterization of mAbs directed against rh IL-2.
Table 2. Determination of specificity of mAbs directed against rh IL-2 using an ELBA.
Data shown represent the isotype of the mAbs directed against rh IL-2 together with their K,, values (n = 6 or 7). K,, values were obtained by performing the method described by Beatty et al.” ID,, values, in the presence of 0.75 III/ml of rh IL-2, final concentrations, were obtained using the CTLL-2 bioassay.
Several lymphokines were used, in concentrations ranging from 1 to 10 &ml, for determination of the specificity of the mAbs directed against rh IL-2. The ELISA was performed as described in Materials and Methods. Positive controls of rh IL-4, rh IFN-y, and TNFo( were performed. -, 4x background.
mAb
AMCIB 21 22 23 24 25 26 27 28 29
ISJtype
W, IiS4 I@-% IgG, kG, IgG, 18-4 IgG, kG,
KaE( x lo-” M) (Mean t SD)
AMCIB
IQ, WW
3.4 f 1.1 14.9 2 5.5 3.5 2 0.5 6.6 2 2.3 1.7 zt 0.8 10.0 ? 3.8 1.8 2 0.5 11.0 * 3.5 9.8 e 6.9
>40 >40 2 9 30 >40 >40 6 >40
This first screening revealed that 164 out of 768 wells were positive. Of these 164 wells, the 96 most positive wells were tested for specific IgG production, and in 49 wells hybridomas producing antibodies of the IgG class were found. This procedure was repeated one week later and 43 wells were found to be double positive for specific IgG antibody production. Of these 43 wells, the 25 most positive were cloned by limiting dilution. This resulted in the growth of 14 stable hybridomas producing specific antibodies of the IgG, class (Table 1). We produced ascites fluid from nine of the stable hybridomas for further experiments. ELISA showed that ascites fluid of all nine hybridomas gave extinctions of at least twice the background values, at lo5 dilution, whereas a mouse mAb specific for human thyroglobulin did not show detectable extinction above the background values, even at lo-fold dilution (data not shown). Experiments using increasing dilutions of an IgG fraction purified as described in Material and Methods revealed the existence of (an) interfering factor(s) that could enhance CTLL-2 proliferation. Therefore, only mAbs purified by affinity chromatography were used for further experiments.
Specificity of Monoclonal Antibodies Directed Against rh IL-2 Specificity for rh IL-2 was investigated in two ways. First, we used an enzyme-linked immunosorbent assay (ELISA), in which several lymphokines were coated directly onto microtiter plates, and, second, we used immunoprecipitation, in which PBMNC were stimulated with PIIA. The different mAbs as tested in the ELISA showed only an interaction with autologous or heterologous IL-2 and there was no detectable interaction with any other of the factors tested (Table 2). Murine anti-(human thyroglobulin) IgG, mAb did not show interaction with any of the factors tested. Control
Coating
rh IL-2 rm IL-2 nh IL-2 PHA-sup rhIL-lP rhIL-4 rhIL-5 &,ILJj rhIFNy rhIFNa, ,.hTN&
21
22
23
24
25
26
27
28
29
AntihTg
++ ++ ++ +++ _ -
++ ++ ++ ++ -
++ + ++ ++ _ _ _ _ _ _
++ + ++ ++ _ _ _ -
++ ++ ++ ++ _ -
++ ++ ++ ++ -
++ ++ ++ +++ -
++ + ++ ++ _ _ -
++ ++ ++ ++ -
-
experiments with mAbs to non-IL-2 cytokines revealed that the coating of these cytokines onto the microtiter plates was sufficient to obtain a signal. In addition, [35S]methionine labeling of PHA stimulated PBMNC followed by immunoprecipitation and polyacrylamide gel electrophoresis (PAGE) revealed the presence of two bands (Fig. 1) with an M, close to
180 84 48 26.5 -
rh IL-2 -
123456789 Figure 1. Determination of specificity of mAbs IL-2 using supernatants from PHA-stimulated labeled with [%I methionine.
c directed PBMNC
against rb that were
PHA-stimulated (24 h) PBMNC were labeled (4 h) with [?S]methionine. The supernatant was immunoprecipitated (overnight, 4°C) using CNBr-activated Sepharose 4B to which the different mAbs, directed against rh IL-2, were coupled. After thoroughly washing, the bound [“Slmethionine labeled nh IL-2 was obtained by incubation of the beads in denaturating buffer for 10 min at 95°C. Twenty microliters of the supernatant were loaded onto a 14% SDSpolyacrylaqmide gel and electrophoresed. Radioactive bands were visualized by autoradiography. Lanes 1-9 represent AMCIB 21-29, respectively, and lane c represents the mAb directed against human thyroglobulin.
56 / Mevissen
CYTOKINE,
et al.
that of rh IL-2. The higher molecular weight band represents the native [35S]-labeled nh IL-2 molecule, whereas the lower band presumably represents degraded nh IL-2. This last observation was also found when rh IL-2 was directly transferred onto nitrocellulose paper and visualized with the different monoclonal antibodies (data not shown).
Afinity ConstantDetermination The affinity constant determination (I
Interleukin 2 (IL-2) is a lymphokine produced by activated human T lymphocytes.‘.’ IL-2 promotes the proliferation of activated T cells, including cytotoxic T cells.3l4In addition, IL-2 promotes the proliferation of other lymphocytes such as natural killer cells,5~6activated B cells,‘x8and lymphokine-activated killer cells.9Y10 IL-2 mediates its growth-promoting effects by interaction with a high-affinity receptor. This high-affinity receptor is composed of at least two different glycoproteins: a low-affinity receptor (Tat-antigen, ~55) and the p7O/p75 antigen.“.l4 In addition, there are indications that the receptor could have a more complex structure. Several authors have identified a number of glycoproteins that are closely associated with the IL-2 receptor.‘5.‘6 The functional role of these glycoproteins remains to be elucidated.
The production of three useful monoclonal antibodies (mAbs) directed against purified native human IL-2 has recently been achieved.” Recombinant DNA technology has made it possible to obtain large amounts of active recombinant human (rh) IL-2. Due to glycosylation, the molecular weight of native human (nh) IL-2 is about 1 kD higher than that of the recombinant form.18 So far, no functional differences are known between recombinant and native human IL-2. In the present study we are the first to report on a group of mAbs raised against rh IL-2. These mAbs recognize both native human IL-2 and rh IL-2 and differ in their ability to inhibit the biological action of the different forms of IL-2.
RESULTS Hybridoma Mono&ma1
‘Laboratory of Cell Biology and Histology and *EC. Slater Institute for Bibchemical R&earch, Acadkmical Medical Center, Meibergdreef 15. 1105 AZ Amsterdam, The Netherlands. ‘Present adhress: Department of Immunology, Cetus Corporation, Emeryville, California, USA. *To whom correspondence should be sent. Copyright 0 1991 by W.B. Saunders Company 1043-4666/91/0301-0005$05.00/O KEY
54
WORDS:
IL-Zimonoclonal
antibodies/structural
differences
and Ascites Fluid Production for Antibodies with Specificity for rh IL-2
Ten days after the second booster, serial dilutions of the serum of one mouse were tested for inhibition of the action of IL-2. We found that increasing concentrations of serum decreased the rh IL-2-dependent proliferation of the CTLL-2 cells (data not shown). Two weeks after fusion, the culture supernatants were tested for specific total immunoglobulin production. CYTOKINE,
Vol. 3, No. 1 (Januaxy),
1991: pp 54-59
Monoclonal antibodies raised against rh IL-2 / 55
Table 1. Characterization of mAbs directed against rh IL-2.
Table 2. Determination of specificity of mAbs directed against rh IL-2 using an ELBA.
Data shown represent the isotype of the mAbs directed against rh IL-2 together with their K,, values (n = 6 or 7). K,, values were obtained by performing the method described by Beatty et al.” ID,, values, in the presence of 0.75 III/ml of rh IL-2, final concentrations, were obtained using the CTLL-2 bioassay.
Several lymphokines were used, in concentrations ranging from 1 to 10 &ml, for determination of the specificity of the mAbs directed against rh IL-2. The ELISA was performed as described in Materials and Methods. Positive controls of rh IL-4, rh IFN-y, and TNFo( were performed. -, 4x background.
mAb
AMCIB 21 22 23 24 25 26 27 28 29
ISJtype
W, IiS4 I@-% IgG, kG, IgG, 18-4 IgG, kG,
KaE( x lo-” M) (Mean t SD)
AMCIB
IQ, WW
3.4 f 1.1 14.9 2 5.5 3.5 2 0.5 6.6 2 2.3 1.7 zt 0.8 10.0 ? 3.8 1.8 2 0.5 11.0 * 3.5 9.8 e 6.9
>40 >40 2 9 30 >40 >40 6 >40
This first screening revealed that 164 out of 768 wells were positive. Of these 164 wells, the 96 most positive wells were tested for specific IgG production, and in 49 wells hybridomas producing antibodies of the IgG class were found. This procedure was repeated one week later and 43 wells were found to be double positive for specific IgG antibody production. Of these 43 wells, the 25 most positive were cloned by limiting dilution. This resulted in the growth of 14 stable hybridomas producing specific antibodies of the IgG, class (Table 1). We produced ascites fluid from nine of the stable hybridomas for further experiments. ELISA showed that ascites fluid of all nine hybridomas gave extinctions of at least twice the background values, at lo5 dilution, whereas a mouse mAb specific for human thyroglobulin did not show detectable extinction above the background values, even at lo-fold dilution (data not shown). Experiments using increasing dilutions of an IgG fraction purified as described in Material and Methods revealed the existence of (an) interfering factor(s) that could enhance CTLL-2 proliferation. Therefore, only mAbs purified by affinity chromatography were used for further experiments.
Specificity of Monoclonal Antibodies Directed Against rh IL-2 Specificity for rh IL-2 was investigated in two ways. First, we used an enzyme-linked immunosorbent assay (ELISA), in which several lymphokines were coated directly onto microtiter plates, and, second, we used immunoprecipitation, in which PBMNC were stimulated with PIIA. The different mAbs as tested in the ELISA showed only an interaction with autologous or heterologous IL-2 and there was no detectable interaction with any other of the factors tested (Table 2). Murine anti-(human thyroglobulin) IgG, mAb did not show interaction with any of the factors tested. Control
Coating
rh IL-2 rm IL-2 nh IL-2 PHA-sup rhIL-lP rhIL-4 rhIL-5 &,ILJj rhIFNy rhIFNa, ,.hTN&
21
22
23
24
25
26
27
28
29
AntihTg
++ ++ ++ +++ _ -
++ ++ ++ ++ -
++ + ++ ++ _ _ _ _ _ _
++ + ++ ++ _ _ _ -
++ ++ ++ ++ _ -
++ ++ ++ ++ -
++ ++ ++ +++ -
++ + ++ ++ _ _ -
++ ++ ++ ++ -
-
experiments with mAbs to non-IL-2 cytokines revealed that the coating of these cytokines onto the microtiter plates was sufficient to obtain a signal. In addition, [35S]methionine labeling of PHA stimulated PBMNC followed by immunoprecipitation and polyacrylamide gel electrophoresis (PAGE) revealed the presence of two bands (Fig. 1) with an M, close to
180 84 48 26.5 -
rh IL-2 -
123456789 Figure 1. Determination of specificity of mAbs IL-2 using supernatants from PHA-stimulated labeled with [%I methionine.
c directed PBMNC
against rb that were
PHA-stimulated (24 h) PBMNC were labeled (4 h) with [?S]methionine. The supernatant was immunoprecipitated (overnight, 4°C) using CNBr-activated Sepharose 4B to which the different mAbs, directed against rh IL-2, were coupled. After thoroughly washing, the bound [“Slmethionine labeled nh IL-2 was obtained by incubation of the beads in denaturating buffer for 10 min at 95°C. Twenty microliters of the supernatant were loaded onto a 14% SDSpolyacrylaqmide gel and electrophoresed. Radioactive bands were visualized by autoradiography. Lanes 1-9 represent AMCIB 21-29, respectively, and lane c represents the mAb directed against human thyroglobulin.
56 / Mevissen
CYTOKINE,
et al.
that of rh IL-2. The higher molecular weight band represents the native [35S]-labeled nh IL-2 molecule, whereas the lower band presumably represents degraded nh IL-2. This last observation was also found when rh IL-2 was directly transferred onto nitrocellulose paper and visualized with the different monoclonal antibodies (data not shown).
Afinity ConstantDetermination The affinity constant determination (I
