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Efficient production of syngeneic anti-IgE monoclonal antibodies with high affinity and diverse specificity
JOURNAL OF IMMUWOLOGICAL METHODS ELSEVTER
Journal
of Immunological
Methods
183 (1995) 199-209
Efficient production of syngeneic anti-IgE monoclonal antibodies with high affinity and diverse specificity Seiji Haba
*,
Alfred Nisonoff
Rosenstiel Research Center, Department of Biology. Bruncleis Unil,ersity. Wulthum, MA 0172.51,USA Received
25 January
1995; accepted
24 February
lYY5
Abstract Syngeneic monoclonal anti-IgE antibodies arc of value in studies of the suppression of IgE synthesis. Procedures are described here for the production of high titers of murine anti-IgE antibodies by initiating immunization in the perinatal period, before mice develop tolerance to their autologous IgE. This in turn facilitates the production of monoclonal anti-IgE antibodies. Properties of some of these mAbs are reported, including affinity, fine specificity and ability to bind to IgE on B lymphoma cells or mast cells.
Keywords:
IgE; Monoclonal
antibody; Anti-IgE hybridoma; Specificity of anti-IgE
1. Introduction Syngeneic monoclonal antibodies against IgE are of value in studies of the mechanism of synthesis and suppression of IgE. The production of high-affinity syngeneic anti-immunoglobulin antibodies is, in general, complicated by immune tolerance to these molecules. (Rheumatoid factors are generally of relatively low affinity.) One
Abbreviations: Ars. p-azophenylarsonate; BSS, balanced salt solution; CAF,, BALB/cXA/J F,; CFA, complete Freund’s adjuvant; D-MEM, Dulbecco’s minimal essential medium; FCS, fetal calf serum; KLH, keyhole limpet hemocyanin; mAb, monoclonal antibody; NMIgG, nonspecific mouse IgG; PVC, polyvinylchloride; RAMFab, rabbit antibodies to mouse Fab. ’ Corresponding author. Tel.: (617) 736-2447; Fax: (617) 736-2405. 0022-1759/95/$09.50 0 1995 Elsevier 759(95)00056-9
SSDI 0022-I
Science
exception to this generalization is IgE. We have shown that tolerance to IgE in adult mice is confined to T cells, and that precursor B cells with anti-IgE specificity are present and readily stimulated by a KLH-IgE conjugate; KLH provides the necessary T cell help (Haba and Nisonoff, 1987a). Furthermore, tolerance at the T cell level only begins at the age of about 2 weeks, when IgE first appears in the mouse (Haba and Nisonoff. 1988). Before this time mice respond to unconjugated syngeneic IgE, and the production of anti-IgE antibodies (with concomitant suppression of IgE synthesis) can be maintained indefinitely by periodic inoculations of IgE (Haba and Nisonoff, 1990). Mean titers of anti-IgE antibodies obtained in this way are considerably greater than those derived when KLH-IgE is used as the immunogen. In addition, the conjugation with KLH undoubtedly interferes sterically with par-
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S. Haba. A. Nisonoff / Journal of Immunological Methods 183 (1995) 199-209
titular regions of the IgE molecule and may alter the repertoire of specificities of anti-IgE mAbs produced. We have taken advantage of this perinatal responsiveness to produce a substantial number of syngeneic anti-IgE mAbs by initiating immunization with unconjugated syngeneic monoclonal IgE at the age of 1 week. The method for generating these mAbs is described here. Some of their properties have also been investigated, including affinity, specificity for regions of the IgE molecule, capacity to bind to IgE on mast cells or B lymphoma cells, and ability to initiate serotonin release and anaphylactic reactions.
2. Materials
and methods
2.1. Mice
A/J and (BALB/c x A/J)F, (CAF,) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). 2.2. Monoclonal antibodies of various isotypes Each mAb was of the K type. IgE mAbs SE1.3, SE20.2 (Haba and Nisonoff, 19851, SE17.1 and 6-16E (Haba and Nisonoff, 1991) are of A/J origin and are specific for p-azobenzenearsonate (Ars). TIB142 and TIB141 (American Type Culture Collection (ATCC), Rockville, MD) are from the BALB/c and C57BL/6 strains, respectively, and have specificity for the trinitrophenyl hapten (Rudolph et al., 1981). Other mAbs, all Arsspecific, are SM1.5 (IgMl (Robbins et al., 1985), SA131 (IgA) (Haba and Nisonoff, 1987a1, R16.7 (IgGl), R22.4 (IgG2a), R9.3 (IgG2b1, and R13.4 (IgG3) (Lamoyi et al., 1980). Each mAb was affinity purified by using a specific hapten for elution, as described elsewhere (Haba et al., 1985). The anti-CD23 mAb, 4B4, was the generous gift of D.H. Conrad. 2.3. Cell lines The FcsRI+ mouse mast cell line, MC/9 (Galli et al., 1982) and the Fc&RII+ (CD23+) B cell
lymphoma, WEHI (Waldschmidt et al., 1988) were obtained from the ATCC. The rat basophilic leukemia line, RBL-2H3 (Barsumian et al., 1981) was the generous gift of F.-T. Liu. 2.4. Immunization of mice and preparation of antiIgE mAbs A/J mice were inoculated i.p. with 100 pg amounts of soluble IgE (6-16El in saline at the age of 7 and 10 days. Thereafter they were injected 4 times with the same amount of IgE in saline at intervals of 1 month. To prepare anti-IgE hybridomas, spleens were removed 5 days after the last immunization for fusion with Sp2/0 cells. Cell fusion was performed as previously described (Haba and Nisonoff, 1987a); hybridoma supernatants were assayed for anti-IgE activity by the solid phase method described below. AntiIgE-producing hybridomas were recloned and grown in ascites in pristane-pretreated CAF, mice. Anti-IgE mAbs were purified from ascites by ammonium sulfate precipitation at 45% of saturation followed by ion-exchange chromatography on a DE52-cellulose column (Whatman, Kent, UK). Some anti-IgE mAbs were affinity purified by using an IgE mAb (SE20.21 coupled to a Sepharose 4B column; they were eluted with 3 M NaSCN. 2.5. Preparation of proteolytic fragments of murine IgE
Proteolytic fragments of SE17.1 and 6-16E were prepared as described previously (Haba and Nisonoff, 1991). F(ab’), fragments were obtained by peptic digestion and Fab fragments by papain digestion. We were unable to obtain intact Fc fragments. 2.6. Solid-phase assay for anti-IgE antibody (Haba and NisonofJ; 1987a) Polyvinylchloride (PVC) microtiter plates were coated with IgE mAb SE20.2, SE17.1 or 6-16E (1 pg/lOO pi/well), and saturated with 0.5% BSA. After exposure to 100 ~1 of test sample for 6 h at
S. Haba, A. Nisonoff /Journal
of Immunological Methods
room temperature, the plates were washed and exposed overnight to 100 ng in 100 Fl/well of ‘2sI-labeled, affinity-purified goat anti-mouse Fc (of IgG). After washing, the radioactivity bound to wells was measured in a gamma counter. To assess the fine specificity of anti-IgE mAb, PVC plates were coated with IgE fragments (Fab or F(ab’&), 1 Kg/100 ~1 per well, and processed as above. 2.7. Isotype specificity of anti-IgE mAbs PVC plates were coated with one of a variety of mAb: IgM, IgGl, IgG2a, IgG2b, IgG3, IgA or IgE; wells were exposed overnight to 1 pg of the coating protein in 100 ~1 borate-buffered saline, pH 8. The plates were then washed and saturated with 0.5% BSA. Wells were exposed to 50 ng/lOO /.ll of ‘251-labeled anti-IgE mAb for 16 h at room temperature and individual wells were counted after washing (Haba and Nisonoff, 1987b). 2.8. Liquid-phase assay for anti-IgE; affinity measurements These assays were carried out by using a fixed amount of anti-IgE and varying amounts of “‘Ilabeled IgE (6-16E) (Haba and Nisonoff, 1987a). Each test mixture also contained, as carrier, 5 ~1 of normal A/J mouse serum which had been adsorbed to remove IgE by passage through a rabbit anti-IgE-Sepharose column. Complexes were precipitated with rabbit anti-mouse Fc (of IgG) which had been preadsorbed with mouse IgE conjugated to Sepharose 4B. After standing overnight, the radioactivity in the precipitate and the supernatant was measured. Each assay mixture also contained 22NaCl; this permitted estimation of the amount of supernatant remaining in the precipitate, so that a washing procedure was not required. The affinity constant for association (K;,) was determined by Scatchard analysis. 2.9. Assay for binding of anti-IgE mAb to IgE + B cells Binding of ‘251-labeled anti-IgE mAb to IgE receptors on B cells was measured by using 6-16E
183
(1995)
199-209
70
1
hybridoma cells, which express membrane-associated IgE. 2 X 10h cells were mixed with 200 ng ‘“51-labeled anti-IgE mAb in 200 ~1 PBS which contained 2% FCS and 500 pg/ml nonspecific mouse IgG (NMIgG). The mixture was incubated on ice with occasional shaking for 90 min. The cells were pelleted after addition of 800 ~1 cold PBS containing 2% FCS and washed once with 1 ml of the same medium; radioactivity in the pellet was then counted. The background level was determined by addition to the mixture of a large excess (100 pg> of unlabeled IgE (6-16E) as an inhibitor, and specific binding was calculated by subtracting background values, which were < 0.3% of the total radioactivity. Assays were carried out in duplicate or, in some instances, in triplicate. 2.10. Binding of “51-labeled anti-&E mAb to I,E bound to Fcs RI Cells of the murine mast cell line MC/9 (5 x 10h cells/ml) were incubated with mAb 6-16E (S pg/ml) at room temperature for 1 h; cells were washed and suspended in PBS containing 2% FCS and 500 pg/ml NMIgG. 200 ng of “‘Ilabeled anti-IgE mAb was then added to 2 X 10” mast cells in a final volume of 200 ~1 and incubated on ice for 90 min; cells were pelleted and washed once. To determine the background level, 100 pg of unlabeled IgE (6-16E) was added to the cell suspension before addition of “51-labeled anti-IgE mAb; this background value was subtracted from experimental values. 2.11. Binding of “51-labeled anti-IgE mAb to IgE bound to Fee RI/ (CD23) on B cells Murine B cell lymphoma (WEHI279) cells (5 10h/ml) were incubated with IgE (6-16E, 10 pg/ml) on ice for 1 h; the cells were washed and suspended in PBS containing 2% FCS and 500 pg/ml NMIgG. 200 ng of “51-labeled anti-IgE mAb was added to 2 x 10h lymphoma cells in 200 ~1 and incubated on ice for 90 min. After centrifuging and washing, the radioactivity in the pellet was measured. The background level was determined by incubating the reaction mixture in the presence of 100 pug of unlabeled IgE. X
202
S. Haba, A. Nisonoff /Journal
of Immunological Methods 183 (1995) 199-209
2.12. Blocking of IgE binding to FCE RI or FceRli by anti-IgE mAbs
To assess the ability of an anti-IgE mAb to block the binding of IgE to Fc&RI on mast cells, varying amounts of anti-IgE mAb were mixed with 200 ng ‘251-labeled IgE (6-16E) in 100 ~1; as a control no anti-IgE was added. After 30 min incubation, lo6 mast cells in 100 ~1 were added and incubated for 90 min on ice. The reaction mixture also contained 2% FCS and 500 pug/ml NMIgG. The mixture was centrifuged after addition of 800 ~1 cold PBS/2.5% FCS; cells were washed once and the radioactivity in the pellet determined. In the absence of inhibitor, 1523% of the total radioactivity was precipitated. When 100 Fg of unlabeled IgE was added as an inhibitor, binding of radioactivity was inhibited by more than 99%. To measure the inhibition of binding of IgE to FcERII (CD23), WEHI B lymphoma cells (2 X lO’/tube) were used instead of mast cells. With no inhibitor present 16-23% of the radioactivity was bound. When 100 pg of unlabeled IgE or 100 pug of the anti-CD23 mAb, 4B4, was used as an inhibitor, > 99% inhibition of binding was observed. 2.13. Degranulation assay Rat basophilic leukemia cells, RBL-2H3, were grown in D-MEM/20% FCS. The cell suspension (lo6 cells/ml) was incubated with 5 pCi/ml (final concentration) [‘t’lhydroxytryptamine (serotonin) and plated in 48 well culture plates (2 x 10” cells/200 ~1 per well). After overnight incubation at 37°C in 5% CO,, the plates were washed 3 x with Hanks’ BSS containing 0.1% BSA. (The cells adhere to plastic.) 200 ~1 medium containing 5 pg/ml IgE mAb (6-16E) was then added to each well. After 1 h incubation at 37” C, the culture plates were washed three times and the cells further cultured in the presence of antiIgE mAb (final concentration, 10 pg/ml) in 200 /*l of Hanks’ BSS, 0.1% BSA, for 90 min at 37” C. 100 ~1 of supernatant was taken from each well for counting of radioactivity. The extent of spontaneous release was measured by adding medium
only. The maximum release of radioactivity was determined by addition of 1% Triton X-100. Assays were done in duplicate. 2.14. Anaphylactic reactions in the mouse paw 3-6-month-old CAF, mice (IgE concentration in serum, l-3 pg/ml) were used. Dilutions of anti-IgE mAb were injected into the hind foot pads (20 pi/site) and an intravenous injection of 0.2 ml of 0.2% Evans Blue dye was given immediately. Observations were made 15 min later. If most or all of the foot pad turned blue, the result was scored as positive (Martel and Khcius, 1977; Kaneta et al., 1984).
3. Results 3.1. Induction of high titers of anti-IgE antibodies and preparation of anti-IgE hybridomas
Four A/J mice were each injected i.p. with 100 pg of IgE mAb (6-16El in saline at the age of 7 and 10 days. Thereafter, the same amount of IgE solution was inoculated monthly; the serum titer of anti-IgE was determined 14 days after each antigenic stimulation. The anti-IgE titer increased significantly after each injection but tended to level off after 4 months; the range of anti-IgE titers was 560-1370 pg/ml with a mean value of 900 pg/ml. This is considerably higher than the average obtained by immunizing adult A/J mice with IgE-KLH in CFA (Haba and Nisonoff, 1987al. Another group of four mice was inoculated six times as above with IgE in saline. Spleens were then removed and single cell suspensions prepared. Recovery of the cells after lysis of RBC was h 4 x 10’ per spleen. No enlargement of spleens or abdominal adhesions were discernible; the latter symptoms are typical of mice receiving repeated inoculations of CFA. From four fusion events, we isolated 26 clones reactive with the immunogen (6-16E). Seven of these clones reacted only with the immunogen (6-16E) but not with other IgE mAbs; they were regarded as anti-idiotypic and not studied fur-
S. Haba, A. Nisonoff /Journal
70.1
of Immunological Method3 183 (19951 199-209
Isotype specificity of these anti-IgE mAbs was ascertained as described in the materials and
methods section, and the results are shown in Table 1. All “‘I-labeled anti-IgE mAbs showed significant binding to IgE-coated wells. For each mAb 13-70% of the radioactivity was bound to the three IgE mAbs tested (TIB142, TIB141 and 616E). There was little or no binding to wells coated with NMIgG or monoclonal IgM, IgGl, IgG2a, IgG2b, IgG3 or IgA. As a positive control, to show that the wells had been adequately coated, each coating was tested with ‘251-labeled rabbit anti-mouse Fab (RAMFab). The results shown in the last column of Table 1 indicate that roughly comparable amounts of Ig were present on the various coated wells; very little labeled RAMFab was bound to wells saturated with BSA but lacking Ig (first row). The anti-IgE mAbs tested showed variability with respect to fine specificity. Although each reacted well with intact IgE only AE19 and AE20
Table 1 Specificity
as “‘I-bound
ther. Nineteen clones showed reactivity with all IgE mAb tested (SE1.3, 6-16E, SE17.1, TIB142, and TIB141). Of these 19 mAbs, 11 were found to be reactive with Fab fragments of IgE and eight were non-reactive. The latter eight mAbs were presumed to be specific for Fc. (Intact Fc fragments of IgE are difficult to prepare and were not available for testing.) Ten mAbs, two of which were reactive with Fab of IgE, were selected for further study. In addition we investigated the properties of a previously isolated mAb, AEll, obtained after immunization with KLHIgE in CFA (Haba and Nisonoff, 1987b). AEll does not react with Fab of IgE. 3.2. Evidence for isotype specificity of anti-IgE mAbs
of monoclonal
Coating of wells a
anti-IgE
‘asI-labeled
antibodies
anti-IgE
(values
are expressed
(cpm)
mAb
““I-labeled RAMFab h
AEll
AE18
AE19
AE20
AE21
AE23
AE33
None ’ NMIgG ’ IgM (SM1.51 ’ IgA (SAl31) IgGl (R16.7) IgG2a (R22.4) IgG2b (R8.2) IgG3 (R13.41 IgE tTIB142. Ig-7a) ’
600 800 1000 600 800 800 700 700 122300
1600 3000 950 1000 1500 1200 900 1000 97900
900 1400 2 200 1500 1 400 1600 1 600 2 000 237700
600 1600 900 500 700 800 700 700 152600
800 1900 900 900 900 900 600 700 188000
400 700 400 400 800 700 600 700 52400
400 700 400 400 700 600 700 600 61300
1800 1900 1800 2 000 2 100 2 100 2 100 2 300 91400
800 1500 1000 900 1600 1800 1700 1200 94200
IgE (TlB141. Ig-7b)
155600
56300
180800
103 100
141400
67700
73900
106 100
IgE (6.16E) F(ab’)? of 6-16E Fab of 6 16E Total cpm added (X 10-V
162100 10100 3 700 306
108200 8800
194200 167300 85 800 420
118100 108100 57000 406
171600 178400 8100 414
91500 800 600 412
217900 800 700 499
180600 5 100 2500 483
1000 383
AE34
AE35
AE36
AE38
600 800 600 600 600 800 700 700 132300
2 300 2 700 2 no0 2 000 7 100 2 200 2500 2500 161500
100500
205700
98400
27 900
141400 1500 2 300 389
157300 3 300 3 600 504
149200 3 400 4 500 538
31000 77 700 23 800 107
“ Wells were coated with protein (1 fig/l00 PI/well) and saturated with BSA as described with ‘asI-labeled anti-IgE (50 ng/well) or Iz I-labeled RAMFab (100 ng/well). ’ Rabbit antibodies against Fab fragments of mouse IgG. ’ Coated with BSA only. ’ Nonspecific mouse IgG. ’ Symbols in parentheses are the names of mAbs. ’ Ig-7a and lg-7b are allotypes of IgE.
in “Methods”:
27 27 27 46 23 20 30 22
400 600 200 500 700 900 700 900 300
they were developed
204
S. Haba. A. Nisonoff /Journal
of Immunological Methods 183 (1995) 199-209
reacted strongly with Fab fragments of IgE. These two mAbs also reacted strongly with F(ab’),. AE21 reacted with F(ab’), but not with Fab. The remaining 8 anti-IgE mAbs reacted poorly with the fragments.
IgE (Table 2, footnote f), indicating that the binding is IgE-specific. The anti-IgE mAbs were highly variable with respect to their capacity to bind to mast cells treated with IgE (Table 2); six of the 11 mAbs were bound. All of the anti-IgE mAbs bound to CD23+ B lymphoma cells (WEHI279) that had been treated with IgE (Table 2); however, the degree of binding of two of the mAbs (AE21 and AE36), although higher than background, was considerably lower than that of the other nine. (AE36 interacted poorly with IgE on mast cells as well as on B lymphoma cells.)
3.3. Affinity The affinity of anti-IgE mAbs for intact IgE in solution was determined by using a fixed amount of anti-IgE and varying amounts of ‘251-labeled (6-16E). The results are shown in Table 2, column 3. The K, values determined by Scatchard analysis varied from 3.6 x 10’ to 1.1 X 10’” M-l; 7 of the 11 have K, values 2 1 X IO” M-‘.
3.5. Inhibition of binding of IgE to mast cells
3.4. Binding of anti-IgE mAbs to IgE on cell surf aces With adhered (6-16E); 2). The presence
the exception of AE21, each of the mAbs to the surface of IgE hybridoma cells 3.6-6.5 ng/lOh cells was bound (Table binding was strongly inhibited in the of a large excess (100 pug) of unlabeled
Table 2 Binding properties Anti-IgE mAb
AEll AE18 AE19 AE20 AE21 AE23 AE33 AE34 AE35 AE36 AE38
The fine specificity of binding of anti-IgE mAbs to IgE was investigated further by measurements of inhibition of the binding of labeled IgE to receptors on mast cells (Table 3). Eight of 11 mAbs strongly inhibited the binding, whereas 3 (AE19, AE20, AE21) inhibited weakly or not at all. (The highest concentration of anti-IgE tested as inhibitor was 500 times greater than that of the
of syngeneic
anti-IgE
Specificity (for IgE or its fragments) a
IgE IgE IgE, F(ab’f,, Fab IgE, F(ab’ jz Fab IgE. F(ab’ j2 IgE IgE IgE IgE IgE IgE
mAbs Affinity for intact IgE h
Binding
(K,,
IgE hybridoma cells ’
Mast cells d (ng/106 cells ‘)
CD23+ B lymphoma ’
5.6 6.5 5.3 3.6 1.4 4.2 4.8 5.5 4.3 5.1 4.3
0 0 11 11 5.4 7.9 5.0 0 2.4 0 0
6.3 6.3 7.7 8.8 1.8 I1 6.6 5.5 7.5 0.7 5.0
Mm’)
4.7x108 1.0x10” 1.2x10y 1.2x10” 2.3~10s 3.6x10’ 1.1x10’” 2.6x10’ 7.3x10” 6.9~10’ 2.3x10”
of “51-labeled
a From Table 1. h Determined in the liquid phase (see materials and methods section). Hybridoma 6-16E, which secretes IgE and expresses IgE on the cell surface. d Murine mast cell line, MC/9. sensitized with 5 pg/ml IgE mAb 6-16E. ’ Cell line WEH1279, sensitized with 10 yg/ml IgE mAb 6-16E. ’ All data are corrected for background values ( < 0.15 ng) obtained by adding “51-anti-IgE.
anti-IgE
to
a large excess of unlabeled
IgE prior to addition
of
S. Haba, A. Nisonqff/Joumal
Table 3 Inhibition by anti-IgE mAbs of binding of ‘251-labeled IgE to mast cells ” (values are expressed as % inhibition of binding of “?-labeled IgEl Concentration
mAb
AEll AE18 AE19 AE20 AE21 AE23 AE33 AE34 AE35 AE36 AE38
of anti-IgE
mAb (pg/ml)
500
5
0.5
0.15
99 99 12 II 11 89 96 99 91 96 98
98 98 NT ’ NT NT 59 8X 97 93 96 96
83 80 NT NT NT 16 66 78 45 70 82
30 36 NT NT NT 3 27 34 15 34 29
205
of Immunological Methods 183 (199.~) 199-209 Table 4 Inhibition by anti-IgE mAb of binding of “‘1-1gE phoma cells a (values are expressed as % inhibition of “‘I-labeled IgE)
h
AJZll AEl8 AE19 AE20 AE21 AE23 AE33 AE34 ‘4E35 AE36 AE38
with a 200 ng of ‘251-labeled IgE mAb 6-16E was incubated varying concentrations of anti-IgE mAb in a volume of 100 ~1. The mixture was added to 1~10~ mast cells (MC/91 in 100 yl and incubated for 90 min before washing. In the absence of inhibitor 30-46 ng of labeled IgE was bound. h Final concentration in the mixture. ’ Not tested.
Concentration
of anti-IgE
500
5
0.5
24 46 6
NT h 35 NT NT NT 69 78 28 97 72 28
NT NT NT NT NT II 0 NT 0 I6 NT
I 30 94 88 38 99 99 40
to B lymof binding
mAb (~g/mll
a The B lymphoma line WEHI (2X 10” cells) was used. The procedure is otherwise the same as that specified in footnote a, Table 3. In the absence of inhibitor 32-46 ng of labeled IgE (6-16E) was bound. b Not tested.
3.4. Inhibition
of binding of IgE to B lymphoma
cells labeled ligand.) Three of the anti-IgE mAbs (AE23, AE33, AE35) were intermediate with respect to inhibitory capacity. The relationship between inhibitory capacity and direct binding to IgE on mast cells (Table 2) will be considered in the Discussion.
Table 5 Degranulation Anti-IgE None ’ NMIgG ’ AEll AE18 AE34 AE36 AE38
mAb
of IgE-sensitized
rat basophils % serotonin 7.7 7.7 6.0 7.0 7.1 7.0 7.0
* * + + f * *
0.2 0.4 0.1 0.8 0.3 0.3 0.3
by anti-IgE
release
b
Four of the anti-IgE mAbs (AE23, AE33, AE35, AE36) strongly inhibited the binding of labeled IgE to B lymphoma cells (Table 4). Five of the mAbs were intermediate in their inhibitory capacity and two (AE19 and AE20) showed little
mAb a Anti-IgE AE23 AE33 AE35 AE21 AE19 AE20
mAb
% serotonin 7.7 29 21 6.6 14 19
release
* 0.6 i3 +2 i_ 0.1 +1 *2
a Rat basophilic leukocytes (RBL-2H3 line), intrinsically labeled with [“Hlserotonin (materials and methods section), were exposed to IgE mAb 6-16E (5 Kg/ml) for 1 h, washed and treated with 200 ~1 of the anti-IgE mAb specified (10 pg/ml) for 90 min at 37” c. b The maximum release of tritium, taken as lOO%, was determined by treatment of control cells with 1% Triton X-100. Experiments were in duplicate. ’ Treated with medium only. ’ Nonspecific mouse IgG.
206
S. Haba. A. Nisonoff /Journal
of Immunological Methods 183 (1995) 199-209
or no inhibitory capacity. The relationship between inhibitory capacity and direct binding to IgE attached to B lymphoma cells (Table 2) will be considered in the discussion section. 3.7. Degranulation of IgE-sensitized rat basophilic Leukocytes and induction of local anaphylaxis by anti-IgE mAbs Four of the 11 anti-IgE mAbs tested (AE19, AE20, AE33 and AE35) caused an extent of degranulation that was significantly greater than that of controls, when exposed to basophilic leukocytes sensitized with IgE (Table 5). Six of 11 mAbs induced immediate local anaphylaxis when inoculated into footpads of CAF, mice (Table 61, whereas the other five mAbs gave negative results. F(ab’), fragments of three of these mAbs were also tested. In each case the results corresponded with those observed with the parent, undigested molecule (two positive, one negative). A summary of properties of the various antiIgE mAbs is shown in Table 7.
4. Discussion We describe here a convenient method for the production of syngeneic anti-IgE mAbs. Its basis is to start the immunization of mice before the age of 2 weeks, using an IgE mAb in saline solution as the immunogen, and to inject this material repeatedly over a period of 4-5 months. This procedure resulted in anti-IgE titers of 500-1300 pg/ml, values high enough to facilitate the production of numerous anti-IgE mAbs. The mean titer is considerably higher than that obtainable by inoculation of a KLH-IgE conjugate into adult mice (Haba and Nisonoff, 1987a). We isolated 26 hybridomas producing anti-IgE mAbs. Seven were idiotype-specific and were not investigated further. Nearly half of our mAbs reacted with Fab fragments of IgE (presumably with the CHl region). This finding contrasts with studies on xenogeneic mAbs against mouse (Baniyash and Eshhar, 1984; Keegan et al., 1991) or human IgE (Hook et al., 1991; Prado et al., 19911, in which the mAbs were predominantly
Fc-directed. We selected ten anti-IgE mAbs for more detailed investigation, together with one from a previous study. Each of these mAbs was shown to be specific for isotypic determinants of IgE and non-reactive with other classes of Ig (Table 1). Their affinities (K,) for IgE ranged from 3.6 X 10’ to 1.1 x 10”’ M-‘; the mean value was about 100 times greater than that obtained with adult recipients, using KLH-IgE in CFA as the immunogen (Haba and Nisonoff, 1987b). We are uncertain as to whether the high affinity resulted from the early immunization of the mice or from the use of IgE in saline, rather than KLH-IgE in CFA. (Adult mice respond very poorly to KLH-IgE in saline.) It may also be relevant that the anti-IgE mAbs obtained by this method are predominantly of the IgGl isotype (18 of 19 clones). IgG2a and IgG2b were the predominant isotypes produced in response to KLH-IgE in CFA (Haba and Nisonoff, 1987b). Such a distinctive isotype distribution may be attributable to the different kinds and/or amounts of lymphokines delivered by helper T cells specific for IgE or for KLH, respectively; this might also influence the maturation of affinity. As noted above, a substantial fraction of the mAbs reacted with the Fab region of IgE. One mAb, AE21, was found to react with F(ab’jz fragments, but not with Fab, of IgE. As the F(ab’), fragment of IgE appears to contain complete CH2 domains (Haba and Nisonoff, 1991), this mAb is probably directed towards CH2. We examined the reactivity of the anti-IgE mAbs with IgE molecules bound to FcgRI or FceRII receptors and observed differential reactivities of the various syngeneic anti-IgE mAbs. The anti-IgE mAbs could be divided into several groups according to their direct binding activity or their capacity to inhibit the binding of IgE to Fee receptors. (Other investigators have shown that human (Helm et al., 1988; Vercelli et al., 1989) and mouse IgE (Nissim et al., 1991; Nissim et al., 1993; Weetall et al., 1990) interact with FcFRI and FcERII through distinct regions of the Fc portion of the IgE molecule.) First, AE21, which appears to be specific for the CH2 domain of IgE, recognizes FcsRI- and
S. Haha. A. Nisonoff /Journal Table 6 Induction Anti-IgE
of local anaphylaxis mAb
ng anti-IgE
100
10
_
_
_
_
NT’ NT NT _
_
NT NT NT _
+ +
+ +
NT + +
+ +
NT -
+
+
+
_
_
+ + + + + -
+ + + + +
+ + + + + _
_
_
+ + _
_
+ _
_
_c _ _ _ _ _
_ _
-
1
” CAF, mice, 3-h-months old, were inoculated in a hindfootpad with 20 ~1 of anti-IgE mAb or NMIgG. The other hind footpad received only diluent (Methods). Control mice received NMIgG in the amounts specified. The mice were immediately inoculated i.v. with 200 ~1 of 0.2% Evans Blue dye in saline. Paws were inspected after 15 min and the mice were killed. ’ Nonspecific mouse IgG. ’ A - or + indicates a negative or a positive (blueing) result, respectively. ’ Not tested.
FcERII-bound IgE but does not inhibit the binding of IgE to either of these receptors. This is obviously consistent with the view that AE21 rec-
Table 7 Summary
of properties
of anti-IgE
Binding sJgE ’
IgE bound to FcsRI ’
IgE bound to FccRII ’
F(ab’1, IgE d
AJZll, AE18, AE34. AE38 AE36 AE23, AE33, AE35 AE21 AJZ19,AE20
+
-
+
_
+ +
_ +
+/+
_
+
+ +
+ +
+ +
” ’ ’ ’
199-209
207
mAbs
Anti-IgE mAb
+
(1995)
ognizes epitopes that are not involved in the binding to either receptor. mAbs in a second group - AEl 1, AE18, AE34 and AE38 - do not recognize IgE bound to FceRI but do react with FceRII-bound IgE. They also inhibit the binding of IgE to FcERI but do not inhibit binding to FceRII. This indicates that these four mAbs interact with epitopes that are directly involved (or sterically blocked) in the binding to FcFRI and that remain exposed when IgE is bound to FcsRII. AE36 represents a third type of specificity. It does not interact with FcERI-bound IgE and interacts very poorly with FcERII-bound IgE, but it can inhibit the binding of IgE to both FcFRI and FceRII. The latter observation suggests that the epitopes involved in binding to FcFRI and FceRII are in close enough proximity so that a single mAb can interfere with the binding of IgE to either receptor. (That nonidentical regions of IgE bind to the two receptors is shown by the patterns of binding of other anti-IgE mAbs.) mAbs AE19 and AE20, which are directed against the Fab of IgE, have, as expected, no competitive effects on the binding of IgE to FcsRI or FceRII. Such differential binding and inhibitory activities of anti-IgE mAbs are consistent with previously reported observations, made with xenogeneic anti-mouse or anti-human IgE, that the binding sites of IgE that interact with FcPRI or FcERII are distinct but are located in close prox-
mAbs ’
mAb inoculated 1000
10000 NMIgG h AEll AElf! AE34 AE36 AE38 AE19 AE20 AE2 I AJS23 AE33 AE35 AE19 Ffab’), AE33 F(ab’1, AE38 Flab’),
by anti-IgE
of Immunological Methods 153
to
JgE present on the surface of a B cell hybridoma. Present on a murine mast cell line. Present on a murine B Jymphoma cell line. Bound to a plastic surface.
of
Fab of JgE d
Inhibition of IgE binding to
In vivo anaphylaxis
FcERJ
FcPRJI
+
_
_
_ _
+ +
+ +
_
_
_ _
_
+ +
+
+
208
S. Haba, A. Nisonoff / Journal of Immunological Methods 183 (1995) 199-209
imity to one another (Baniyash and Eshhar, 1984; Baniyash et al., 1988; Davis et al., 1993; Hook et al., 1991; Rings et al., 1986). Another type of specificity is associated with mAbs AE23, AE33 and AE35. Each of these mAbs were bound to IgE attached to either FcsRI or FcsRII. However, in contrast to AE19, AE20 and AE21 (which also interacted with IgE bound to either receptor) AE23, AE33 and AE3.5 were able to inhibit the binding of IgE to each receptor. A possible explanation is that the determinants on IgE with which these mAbs interact are repeated on the IgE molecule in such a way that at least one determinant remains exposed when the IgE bound to a receptor. This interpretation requires the additional assumption that the affinities of the mAbs are such that they can prevent binding when added to the IgE before exposure to the cells but displace bound IgE very slowly or not at all. There is an excellent correlation (Tables 6 and 71 between the capacity of each of the mAbs tested to interact with IgE bound to FceRI and their ability to cause anaphylactic reactions in vivo (in the paw of the mouse). Other investigators have used xenogeneic antiIgE antibodies to modulate IgE antibody production in vivo (Bozelka et al., 1982,1985; Davis et al., 1993; Dessein et al., 1981). A possible drawback for the use of xenogeneic Ig for suppression of IgE synthesis is its immunogenicity. Our studies have focused on the suppression of IgE production by syngeneic monoclonal or polyclonal anti-IgE antibodies (Haba and Nisonoff, 1990,1994a,1994b). From a practical standpoint, the fine specificity of these anti-IgE antibodies may be of great importance for the avoidance of anaphylactic reactions during suppression. The anti-IgE mAbs described here may be useful for the establishment of a mouse model for effective suppression of IgE in humans. In this context, Davis et al. (1991) have described mouse mAbs to human IgE that are specific for membraneanchoring segments of IgE heavy chains present on IgE-producing B cells. They did not react with IgE in solution or with IgE bound to FcERI or FcsRII. Antibodies with appropriate characteristics may prove to be therapeutically useful.
Acknowledgements
This work was supported by Grant IM-607C from the American Cancer Society. We are grateful to Ms. T. Haba for technical assistance.
References Baniyash. M. and Eshhar, Z. (1984) Inhibition of IgE binding to mast cells and basophils by monoclonal antibodies to murine IgE. Eur. J. Immunol. 14, 799. Baniyash, M., Kehry. M. and Eshhar, Z. (1988) Anti-IgE monoclonal antibodies directed at the Fcs receptor binding site. Mol. Immunol. 25, 705. Barsumian, E.L., Isersky, C.. Petrino, M.G. and Siraganian, R.P. (1981) IgE-induced histamine release from a rat basophilic leukemia cell line: Isolation of releasing and non-releasing clones. Eur. J. Immunol. 11, 317. Bozelka, B.E.. McCants, M., Salvaggio, J.E. and Lehrer, S.B. (1985) Effects of anti-E on total and specific IgE levels in adult mice. Int. Arch. Allergy Appl. Immunology 78. 51. Bozelka, B.E., McCants, M.L., Salvaggio. J.E. and Lehrer. S.B. (1982) IgE isotype suppression in anti-E-treated mice. Immunology 46, 527. Davis, F.M., Gossett, L.M., Pinkston, K.L., Liou, R.S., Sun, L.K., Kim, Y.W., Chang, N.T., Chang, T.W.. Wagner, K., Bews, J., Brinkmann, V., Towbin, H., Subramanian, N. and Heusser, C. (1993) Can anti-IgE be used to treat allergy? Springer Semin. Immunopathol. 15. 51. Davis, F.M., Gossett, L.A. and Chang, T.W. (1991) An epitope on membrane-bound but not secreted IgE: Implications in isotype-specific regulation. Biotechnology 9, 53. Dessein, A.J., Parker, W.L., James, S.L. and David, J.R. (1981) IgE antibody and resistance to infection. I. Selective suppression of the IgE antibody response in rats diminishes the resistance and eosinophil response to Trichinella spiralis infection. J. Exp. Med. 153, 423. Galli, S.I., Dvorak, A.M., Marcum, J.H., Ishizaka, T.. Nabel, G., Simonian. H.D., Pyne, K., Goldin, J.M., Rosenberg, R.D., Cantor, H. and Dvorak, H.F. (1982) Mast cell clones: A model for the analysis of cellular maturation. J. Cell. Biol. 95, 435. Haba, S. and Nisonoff, A. (1985) Quantitation of IgE antibodies by radioimmunoassay in the presence of high concentration of non-IgE antibodies of the same specificity. J. Immunol. Methods 85, 39. Haba, S. and Nisonoff, A. (1987a) Induction of high titers of anti-IgE by immunization of inbred mice with syngeneic IgE. Proc. Natl. Acad. Sci. USA 84, 5009. Haba. S. and Nisonoff. A. (1987b) Production of syngeneic autoreactive monoclonal antibodies specific for isotypic determinants of IgE. J. Immunol. Methods 105, 193. Haba, S. and Nisonoff, A. (1988) Immunologic responsiveness of neonatal A/J mice to isotypic determinants of syngeneic IgE. J. Exp. Med. 168, 713.
S. Haha. A. Nisonoff /Journal
of Immunological Methods 183 (1995) 199-209
Haba, S. and Nisonoff, A. (1990) Inhibition of IgE synthesis by anti-IgE: role in long-term inhibition of IgE synthesis by neonatally administered soluble IgE. Proc. Natl. Acad. Sci. USA 87, 3363. Haba, S. and Nisonoff. A. (1991) Proteolytic digestion of mouse IgE. J. Immunol. Methods 138, 15. Haba, S. and Nisonoff, A. (1994a) Role of antibody and T cells in the long-term inhibition of IgE synthesis. Proc. Nat]. Acad. Sci. USA 91, 604. Haba, S. and Nisonoff, A. (1994b) Effects of syngeneic anti-IgE antibodies on the development of IgE memory and on the secondary IgE response. J. Immunol. 152, 51. Haba, S., Ovary, Z. and Nisonoff, A. (19851 Clearance of IgE from serum of normal and hybridoma-bearing mice. J. Immunol. 134. 3291. Helm, B.. Marsh, P.. Vercelli, D., Padlan, E., Gould, H. and Geha. R. (19881 The mast cell binding site on human immunoglobulin E. Nature 331, 180. Hook, W.A., Zinsser, F.V.. Berenstein, E.H. and Siraganian. R.P. (1991) Monoclonal antibodies defining epitopes on human IgE. Mol. Immunol. 28, 631. Kaneta, S.. Ishizuki. S., Fujihara, E. and Mitsuya, M. (19841 Mouse paw anaphylaxis. J. Immunol. Methods 71. 83. Keegan. A.D., Fratazzi, C., Shopes, B., Baird, B. and Conrad. D.H. (19911 Characterization of new rat anti-mouse IgE monoclonals and their use along with chimeric IgE to further define the site that interacts with FcsRII and FceRI. Mol. Immunol. 28. 1149. Kings, M.A., Conroy, M.C., Stadler. B.M., Magnusson, C.G., Skvarii, F. and deWeck, A.L. (1986) Histamine release from human leukocytes by anti-IgE antibodies: influence of multiple or single epitope recognition. Diagn. Immunol. 4, 89. Lamoyi. E., Estess, P., Capra. J.D. and Nisonoff, A. (1980) Heterogeneity of an intrastrain cross-reactive idiotype as-
209
sociated with anti-p-azophenylarsonate antibodies of A/J mice. J. Immunol. 124, 2834. Martel, R.R. and Klicius, J. (1977) Reagin-mediated hypersensitivity reactions in the rat hind paw and its inhibition by anti-anaphylactic drugs. Int. Arch. Allergy Appl. Immunot. 54, 205. Nissim, A., Jouvin. M.-H. and Eshhar, Z. ( I991 ) Mapping of the high affinity Fcs receptor binding site to the third constant region domain of IgE. EMBO. J. 10. 101. Nissim, A., Schwarzbaum, S., Siraganian, R. and Eshhar, Z. (19931 Fine specificity of the IgE interaction with the tow and high affinity Fc receptor. J. Immunol. 150. 1365. Prado, J.M.D.. Jimeno, L.. Obispo, T. and Carreira, J. (19911 Monoclonal antibodies against human IgE. Identification of an epitope sharing properties with the high-affinity receptor binding site. Mol. Immunol. 28, 839. Robbins, P.F., Rosen. E.M., Haba. S. and Nisonoff, A. (19851 Relationship of V, and V, genes encoding three idiotypic families of anti-p-azobenzenearsonate antibodies, Proc. Natl. Acad. Sci. USA 83. 39. Rudolph, A.K., Burrows, P.D. and Wabl, M.R. (19811 Thirteen hybridomas secreting hapten-specific immunoglobutin E from mice with Ig” or Igh heavy chain haplotype. Eur. J. Immunol. 11, 527. Vercelti, D., Helm, B.. Marsh, P., Padlan, E., Geha, R.S. and Gould, H. (19891 The B-cell binding site on human immunoglobulin E. Nature 338, 649. Waldschmidt, T.J., Conrad, D.H. and Lynch, R.G. (1988) The expression of B cell surface receptors. I. The ontogeny and distribution of the murine B cell IgE Fc receptors. J. Immunot. 140, 2148. Weetall, M., Shopes, B., Holowka, D. and Baird, B. (19901 Mapping the site of interaction between murine IgE and its high affinity receptor with chimeric Ig. J. Immunol. 145. 3849.
Journal
of Immunological
Methods
183 (1995) 199-209
Efficient production of syngeneic anti-IgE monoclonal antibodies with high affinity and diverse specificity Seiji Haba
*,
Alfred Nisonoff
Rosenstiel Research Center, Department of Biology. Bruncleis Unil,ersity. Wulthum, MA 0172.51,USA Received
25 January
1995; accepted
24 February
lYY5
Abstract Syngeneic monoclonal anti-IgE antibodies arc of value in studies of the suppression of IgE synthesis. Procedures are described here for the production of high titers of murine anti-IgE antibodies by initiating immunization in the perinatal period, before mice develop tolerance to their autologous IgE. This in turn facilitates the production of monoclonal anti-IgE antibodies. Properties of some of these mAbs are reported, including affinity, fine specificity and ability to bind to IgE on B lymphoma cells or mast cells.
Keywords:
IgE; Monoclonal
antibody; Anti-IgE hybridoma; Specificity of anti-IgE
1. Introduction Syngeneic monoclonal antibodies against IgE are of value in studies of the mechanism of synthesis and suppression of IgE. The production of high-affinity syngeneic anti-immunoglobulin antibodies is, in general, complicated by immune tolerance to these molecules. (Rheumatoid factors are generally of relatively low affinity.) One
Abbreviations: Ars. p-azophenylarsonate; BSS, balanced salt solution; CAF,, BALB/cXA/J F,; CFA, complete Freund’s adjuvant; D-MEM, Dulbecco’s minimal essential medium; FCS, fetal calf serum; KLH, keyhole limpet hemocyanin; mAb, monoclonal antibody; NMIgG, nonspecific mouse IgG; PVC, polyvinylchloride; RAMFab, rabbit antibodies to mouse Fab. ’ Corresponding author. Tel.: (617) 736-2447; Fax: (617) 736-2405. 0022-1759/95/$09.50 0 1995 Elsevier 759(95)00056-9
SSDI 0022-I
Science
exception to this generalization is IgE. We have shown that tolerance to IgE in adult mice is confined to T cells, and that precursor B cells with anti-IgE specificity are present and readily stimulated by a KLH-IgE conjugate; KLH provides the necessary T cell help (Haba and Nisonoff, 1987a). Furthermore, tolerance at the T cell level only begins at the age of about 2 weeks, when IgE first appears in the mouse (Haba and Nisonoff. 1988). Before this time mice respond to unconjugated syngeneic IgE, and the production of anti-IgE antibodies (with concomitant suppression of IgE synthesis) can be maintained indefinitely by periodic inoculations of IgE (Haba and Nisonoff, 1990). Mean titers of anti-IgE antibodies obtained in this way are considerably greater than those derived when KLH-IgE is used as the immunogen. In addition, the conjugation with KLH undoubtedly interferes sterically with par-
B.V. All rights reserved
200
S. Haba. A. Nisonoff / Journal of Immunological Methods 183 (1995) 199-209
titular regions of the IgE molecule and may alter the repertoire of specificities of anti-IgE mAbs produced. We have taken advantage of this perinatal responsiveness to produce a substantial number of syngeneic anti-IgE mAbs by initiating immunization with unconjugated syngeneic monoclonal IgE at the age of 1 week. The method for generating these mAbs is described here. Some of their properties have also been investigated, including affinity, specificity for regions of the IgE molecule, capacity to bind to IgE on mast cells or B lymphoma cells, and ability to initiate serotonin release and anaphylactic reactions.
2. Materials
and methods
2.1. Mice
A/J and (BALB/c x A/J)F, (CAF,) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). 2.2. Monoclonal antibodies of various isotypes Each mAb was of the K type. IgE mAbs SE1.3, SE20.2 (Haba and Nisonoff, 19851, SE17.1 and 6-16E (Haba and Nisonoff, 1991) are of A/J origin and are specific for p-azobenzenearsonate (Ars). TIB142 and TIB141 (American Type Culture Collection (ATCC), Rockville, MD) are from the BALB/c and C57BL/6 strains, respectively, and have specificity for the trinitrophenyl hapten (Rudolph et al., 1981). Other mAbs, all Arsspecific, are SM1.5 (IgMl (Robbins et al., 1985), SA131 (IgA) (Haba and Nisonoff, 1987a1, R16.7 (IgGl), R22.4 (IgG2a), R9.3 (IgG2b1, and R13.4 (IgG3) (Lamoyi et al., 1980). Each mAb was affinity purified by using a specific hapten for elution, as described elsewhere (Haba et al., 1985). The anti-CD23 mAb, 4B4, was the generous gift of D.H. Conrad. 2.3. Cell lines The FcsRI+ mouse mast cell line, MC/9 (Galli et al., 1982) and the Fc&RII+ (CD23+) B cell
lymphoma, WEHI (Waldschmidt et al., 1988) were obtained from the ATCC. The rat basophilic leukemia line, RBL-2H3 (Barsumian et al., 1981) was the generous gift of F.-T. Liu. 2.4. Immunization of mice and preparation of antiIgE mAbs A/J mice were inoculated i.p. with 100 pg amounts of soluble IgE (6-16El in saline at the age of 7 and 10 days. Thereafter they were injected 4 times with the same amount of IgE in saline at intervals of 1 month. To prepare anti-IgE hybridomas, spleens were removed 5 days after the last immunization for fusion with Sp2/0 cells. Cell fusion was performed as previously described (Haba and Nisonoff, 1987a); hybridoma supernatants were assayed for anti-IgE activity by the solid phase method described below. AntiIgE-producing hybridomas were recloned and grown in ascites in pristane-pretreated CAF, mice. Anti-IgE mAbs were purified from ascites by ammonium sulfate precipitation at 45% of saturation followed by ion-exchange chromatography on a DE52-cellulose column (Whatman, Kent, UK). Some anti-IgE mAbs were affinity purified by using an IgE mAb (SE20.21 coupled to a Sepharose 4B column; they were eluted with 3 M NaSCN. 2.5. Preparation of proteolytic fragments of murine IgE
Proteolytic fragments of SE17.1 and 6-16E were prepared as described previously (Haba and Nisonoff, 1991). F(ab’), fragments were obtained by peptic digestion and Fab fragments by papain digestion. We were unable to obtain intact Fc fragments. 2.6. Solid-phase assay for anti-IgE antibody (Haba and NisonofJ; 1987a) Polyvinylchloride (PVC) microtiter plates were coated with IgE mAb SE20.2, SE17.1 or 6-16E (1 pg/lOO pi/well), and saturated with 0.5% BSA. After exposure to 100 ~1 of test sample for 6 h at
S. Haba, A. Nisonoff /Journal
of Immunological Methods
room temperature, the plates were washed and exposed overnight to 100 ng in 100 Fl/well of ‘2sI-labeled, affinity-purified goat anti-mouse Fc (of IgG). After washing, the radioactivity bound to wells was measured in a gamma counter. To assess the fine specificity of anti-IgE mAb, PVC plates were coated with IgE fragments (Fab or F(ab’&), 1 Kg/100 ~1 per well, and processed as above. 2.7. Isotype specificity of anti-IgE mAbs PVC plates were coated with one of a variety of mAb: IgM, IgGl, IgG2a, IgG2b, IgG3, IgA or IgE; wells were exposed overnight to 1 pg of the coating protein in 100 ~1 borate-buffered saline, pH 8. The plates were then washed and saturated with 0.5% BSA. Wells were exposed to 50 ng/lOO /.ll of ‘251-labeled anti-IgE mAb for 16 h at room temperature and individual wells were counted after washing (Haba and Nisonoff, 1987b). 2.8. Liquid-phase assay for anti-IgE; affinity measurements These assays were carried out by using a fixed amount of anti-IgE and varying amounts of “‘Ilabeled IgE (6-16E) (Haba and Nisonoff, 1987a). Each test mixture also contained, as carrier, 5 ~1 of normal A/J mouse serum which had been adsorbed to remove IgE by passage through a rabbit anti-IgE-Sepharose column. Complexes were precipitated with rabbit anti-mouse Fc (of IgG) which had been preadsorbed with mouse IgE conjugated to Sepharose 4B. After standing overnight, the radioactivity in the precipitate and the supernatant was measured. Each assay mixture also contained 22NaCl; this permitted estimation of the amount of supernatant remaining in the precipitate, so that a washing procedure was not required. The affinity constant for association (K;,) was determined by Scatchard analysis. 2.9. Assay for binding of anti-IgE mAb to IgE + B cells Binding of ‘251-labeled anti-IgE mAb to IgE receptors on B cells was measured by using 6-16E
183
(1995)
199-209
70
1
hybridoma cells, which express membrane-associated IgE. 2 X 10h cells were mixed with 200 ng ‘“51-labeled anti-IgE mAb in 200 ~1 PBS which contained 2% FCS and 500 pg/ml nonspecific mouse IgG (NMIgG). The mixture was incubated on ice with occasional shaking for 90 min. The cells were pelleted after addition of 800 ~1 cold PBS containing 2% FCS and washed once with 1 ml of the same medium; radioactivity in the pellet was then counted. The background level was determined by addition to the mixture of a large excess (100 pg> of unlabeled IgE (6-16E) as an inhibitor, and specific binding was calculated by subtracting background values, which were < 0.3% of the total radioactivity. Assays were carried out in duplicate or, in some instances, in triplicate. 2.10. Binding of “51-labeled anti-&E mAb to I,E bound to Fcs RI Cells of the murine mast cell line MC/9 (5 x 10h cells/ml) were incubated with mAb 6-16E (S pg/ml) at room temperature for 1 h; cells were washed and suspended in PBS containing 2% FCS and 500 pg/ml NMIgG. 200 ng of “‘Ilabeled anti-IgE mAb was then added to 2 X 10” mast cells in a final volume of 200 ~1 and incubated on ice for 90 min; cells were pelleted and washed once. To determine the background level, 100 pg of unlabeled IgE (6-16E) was added to the cell suspension before addition of “51-labeled anti-IgE mAb; this background value was subtracted from experimental values. 2.11. Binding of “51-labeled anti-IgE mAb to IgE bound to Fee RI/ (CD23) on B cells Murine B cell lymphoma (WEHI279) cells (5 10h/ml) were incubated with IgE (6-16E, 10 pg/ml) on ice for 1 h; the cells were washed and suspended in PBS containing 2% FCS and 500 pg/ml NMIgG. 200 ng of “51-labeled anti-IgE mAb was added to 2 x 10h lymphoma cells in 200 ~1 and incubated on ice for 90 min. After centrifuging and washing, the radioactivity in the pellet was measured. The background level was determined by incubating the reaction mixture in the presence of 100 pug of unlabeled IgE. X
202
S. Haba, A. Nisonoff /Journal
of Immunological Methods 183 (1995) 199-209
2.12. Blocking of IgE binding to FCE RI or FceRli by anti-IgE mAbs
To assess the ability of an anti-IgE mAb to block the binding of IgE to Fc&RI on mast cells, varying amounts of anti-IgE mAb were mixed with 200 ng ‘251-labeled IgE (6-16E) in 100 ~1; as a control no anti-IgE was added. After 30 min incubation, lo6 mast cells in 100 ~1 were added and incubated for 90 min on ice. The reaction mixture also contained 2% FCS and 500 pug/ml NMIgG. The mixture was centrifuged after addition of 800 ~1 cold PBS/2.5% FCS; cells were washed once and the radioactivity in the pellet determined. In the absence of inhibitor, 1523% of the total radioactivity was precipitated. When 100 Fg of unlabeled IgE was added as an inhibitor, binding of radioactivity was inhibited by more than 99%. To measure the inhibition of binding of IgE to FcERII (CD23), WEHI B lymphoma cells (2 X lO’/tube) were used instead of mast cells. With no inhibitor present 16-23% of the radioactivity was bound. When 100 pg of unlabeled IgE or 100 pug of the anti-CD23 mAb, 4B4, was used as an inhibitor, > 99% inhibition of binding was observed. 2.13. Degranulation assay Rat basophilic leukemia cells, RBL-2H3, were grown in D-MEM/20% FCS. The cell suspension (lo6 cells/ml) was incubated with 5 pCi/ml (final concentration) [‘t’lhydroxytryptamine (serotonin) and plated in 48 well culture plates (2 x 10” cells/200 ~1 per well). After overnight incubation at 37°C in 5% CO,, the plates were washed 3 x with Hanks’ BSS containing 0.1% BSA. (The cells adhere to plastic.) 200 ~1 medium containing 5 pg/ml IgE mAb (6-16E) was then added to each well. After 1 h incubation at 37” C, the culture plates were washed three times and the cells further cultured in the presence of antiIgE mAb (final concentration, 10 pg/ml) in 200 /*l of Hanks’ BSS, 0.1% BSA, for 90 min at 37” C. 100 ~1 of supernatant was taken from each well for counting of radioactivity. The extent of spontaneous release was measured by adding medium
only. The maximum release of radioactivity was determined by addition of 1% Triton X-100. Assays were done in duplicate. 2.14. Anaphylactic reactions in the mouse paw 3-6-month-old CAF, mice (IgE concentration in serum, l-3 pg/ml) were used. Dilutions of anti-IgE mAb were injected into the hind foot pads (20 pi/site) and an intravenous injection of 0.2 ml of 0.2% Evans Blue dye was given immediately. Observations were made 15 min later. If most or all of the foot pad turned blue, the result was scored as positive (Martel and Khcius, 1977; Kaneta et al., 1984).
3. Results 3.1. Induction of high titers of anti-IgE antibodies and preparation of anti-IgE hybridomas
Four A/J mice were each injected i.p. with 100 pg of IgE mAb (6-16El in saline at the age of 7 and 10 days. Thereafter, the same amount of IgE solution was inoculated monthly; the serum titer of anti-IgE was determined 14 days after each antigenic stimulation. The anti-IgE titer increased significantly after each injection but tended to level off after 4 months; the range of anti-IgE titers was 560-1370 pg/ml with a mean value of 900 pg/ml. This is considerably higher than the average obtained by immunizing adult A/J mice with IgE-KLH in CFA (Haba and Nisonoff, 1987al. Another group of four mice was inoculated six times as above with IgE in saline. Spleens were then removed and single cell suspensions prepared. Recovery of the cells after lysis of RBC was h 4 x 10’ per spleen. No enlargement of spleens or abdominal adhesions were discernible; the latter symptoms are typical of mice receiving repeated inoculations of CFA. From four fusion events, we isolated 26 clones reactive with the immunogen (6-16E). Seven of these clones reacted only with the immunogen (6-16E) but not with other IgE mAbs; they were regarded as anti-idiotypic and not studied fur-
S. Haba, A. Nisonoff /Journal
70.1
of Immunological Method3 183 (19951 199-209
Isotype specificity of these anti-IgE mAbs was ascertained as described in the materials and
methods section, and the results are shown in Table 1. All “‘I-labeled anti-IgE mAbs showed significant binding to IgE-coated wells. For each mAb 13-70% of the radioactivity was bound to the three IgE mAbs tested (TIB142, TIB141 and 616E). There was little or no binding to wells coated with NMIgG or monoclonal IgM, IgGl, IgG2a, IgG2b, IgG3 or IgA. As a positive control, to show that the wells had been adequately coated, each coating was tested with ‘251-labeled rabbit anti-mouse Fab (RAMFab). The results shown in the last column of Table 1 indicate that roughly comparable amounts of Ig were present on the various coated wells; very little labeled RAMFab was bound to wells saturated with BSA but lacking Ig (first row). The anti-IgE mAbs tested showed variability with respect to fine specificity. Although each reacted well with intact IgE only AE19 and AE20
Table 1 Specificity
as “‘I-bound
ther. Nineteen clones showed reactivity with all IgE mAb tested (SE1.3, 6-16E, SE17.1, TIB142, and TIB141). Of these 19 mAbs, 11 were found to be reactive with Fab fragments of IgE and eight were non-reactive. The latter eight mAbs were presumed to be specific for Fc. (Intact Fc fragments of IgE are difficult to prepare and were not available for testing.) Ten mAbs, two of which were reactive with Fab of IgE, were selected for further study. In addition we investigated the properties of a previously isolated mAb, AEll, obtained after immunization with KLHIgE in CFA (Haba and Nisonoff, 1987b). AEll does not react with Fab of IgE. 3.2. Evidence for isotype specificity of anti-IgE mAbs
of monoclonal
Coating of wells a
anti-IgE
‘asI-labeled
antibodies
anti-IgE
(values
are expressed
(cpm)
mAb
““I-labeled RAMFab h
AEll
AE18
AE19
AE20
AE21
AE23
AE33
None ’ NMIgG ’ IgM (SM1.51 ’ IgA (SAl31) IgGl (R16.7) IgG2a (R22.4) IgG2b (R8.2) IgG3 (R13.41 IgE tTIB142. Ig-7a) ’
600 800 1000 600 800 800 700 700 122300
1600 3000 950 1000 1500 1200 900 1000 97900
900 1400 2 200 1500 1 400 1600 1 600 2 000 237700
600 1600 900 500 700 800 700 700 152600
800 1900 900 900 900 900 600 700 188000
400 700 400 400 800 700 600 700 52400
400 700 400 400 700 600 700 600 61300
1800 1900 1800 2 000 2 100 2 100 2 100 2 300 91400
800 1500 1000 900 1600 1800 1700 1200 94200
IgE (TlB141. Ig-7b)
155600
56300
180800
103 100
141400
67700
73900
106 100
IgE (6.16E) F(ab’)? of 6-16E Fab of 6 16E Total cpm added (X 10-V
162100 10100 3 700 306
108200 8800
194200 167300 85 800 420
118100 108100 57000 406
171600 178400 8100 414
91500 800 600 412
217900 800 700 499
180600 5 100 2500 483
1000 383
AE34
AE35
AE36
AE38
600 800 600 600 600 800 700 700 132300
2 300 2 700 2 no0 2 000 7 100 2 200 2500 2500 161500
100500
205700
98400
27 900
141400 1500 2 300 389
157300 3 300 3 600 504
149200 3 400 4 500 538
31000 77 700 23 800 107
“ Wells were coated with protein (1 fig/l00 PI/well) and saturated with BSA as described with ‘asI-labeled anti-IgE (50 ng/well) or Iz I-labeled RAMFab (100 ng/well). ’ Rabbit antibodies against Fab fragments of mouse IgG. ’ Coated with BSA only. ’ Nonspecific mouse IgG. ’ Symbols in parentheses are the names of mAbs. ’ Ig-7a and lg-7b are allotypes of IgE.
in “Methods”:
27 27 27 46 23 20 30 22
400 600 200 500 700 900 700 900 300
they were developed
204
S. Haba. A. Nisonoff /Journal
of Immunological Methods 183 (1995) 199-209
reacted strongly with Fab fragments of IgE. These two mAbs also reacted strongly with F(ab’),. AE21 reacted with F(ab’), but not with Fab. The remaining 8 anti-IgE mAbs reacted poorly with the fragments.
IgE (Table 2, footnote f), indicating that the binding is IgE-specific. The anti-IgE mAbs were highly variable with respect to their capacity to bind to mast cells treated with IgE (Table 2); six of the 11 mAbs were bound. All of the anti-IgE mAbs bound to CD23+ B lymphoma cells (WEHI279) that had been treated with IgE (Table 2); however, the degree of binding of two of the mAbs (AE21 and AE36), although higher than background, was considerably lower than that of the other nine. (AE36 interacted poorly with IgE on mast cells as well as on B lymphoma cells.)
3.3. Affinity The affinity of anti-IgE mAbs for intact IgE in solution was determined by using a fixed amount of anti-IgE and varying amounts of ‘251-labeled (6-16E). The results are shown in Table 2, column 3. The K, values determined by Scatchard analysis varied from 3.6 x 10’ to 1.1 X 10’” M-l; 7 of the 11 have K, values 2 1 X IO” M-‘.
3.5. Inhibition of binding of IgE to mast cells
3.4. Binding of anti-IgE mAbs to IgE on cell surf aces With adhered (6-16E); 2). The presence
the exception of AE21, each of the mAbs to the surface of IgE hybridoma cells 3.6-6.5 ng/lOh cells was bound (Table binding was strongly inhibited in the of a large excess (100 pug) of unlabeled
Table 2 Binding properties Anti-IgE mAb
AEll AE18 AE19 AE20 AE21 AE23 AE33 AE34 AE35 AE36 AE38
The fine specificity of binding of anti-IgE mAbs to IgE was investigated further by measurements of inhibition of the binding of labeled IgE to receptors on mast cells (Table 3). Eight of 11 mAbs strongly inhibited the binding, whereas 3 (AE19, AE20, AE21) inhibited weakly or not at all. (The highest concentration of anti-IgE tested as inhibitor was 500 times greater than that of the
of syngeneic
anti-IgE
Specificity (for IgE or its fragments) a
IgE IgE IgE, F(ab’f,, Fab IgE, F(ab’ jz Fab IgE. F(ab’ j2 IgE IgE IgE IgE IgE IgE
mAbs Affinity for intact IgE h
Binding
(K,,
IgE hybridoma cells ’
Mast cells d (ng/106 cells ‘)
CD23+ B lymphoma ’
5.6 6.5 5.3 3.6 1.4 4.2 4.8 5.5 4.3 5.1 4.3
0 0 11 11 5.4 7.9 5.0 0 2.4 0 0
6.3 6.3 7.7 8.8 1.8 I1 6.6 5.5 7.5 0.7 5.0
Mm’)
4.7x108 1.0x10” 1.2x10y 1.2x10” 2.3~10s 3.6x10’ 1.1x10’” 2.6x10’ 7.3x10” 6.9~10’ 2.3x10”
of “51-labeled
a From Table 1. h Determined in the liquid phase (see materials and methods section). Hybridoma 6-16E, which secretes IgE and expresses IgE on the cell surface. d Murine mast cell line, MC/9. sensitized with 5 pg/ml IgE mAb 6-16E. ’ Cell line WEH1279, sensitized with 10 yg/ml IgE mAb 6-16E. ’ All data are corrected for background values ( < 0.15 ng) obtained by adding “51-anti-IgE.
anti-IgE
to
a large excess of unlabeled
IgE prior to addition
of
S. Haba, A. Nisonqff/Joumal
Table 3 Inhibition by anti-IgE mAbs of binding of ‘251-labeled IgE to mast cells ” (values are expressed as % inhibition of binding of “?-labeled IgEl Concentration
mAb
AEll AE18 AE19 AE20 AE21 AE23 AE33 AE34 AE35 AE36 AE38
of anti-IgE
mAb (pg/ml)
500
5
0.5
0.15
99 99 12 II 11 89 96 99 91 96 98
98 98 NT ’ NT NT 59 8X 97 93 96 96
83 80 NT NT NT 16 66 78 45 70 82
30 36 NT NT NT 3 27 34 15 34 29
205
of Immunological Methods 183 (199.~) 199-209 Table 4 Inhibition by anti-IgE mAb of binding of “‘1-1gE phoma cells a (values are expressed as % inhibition of “‘I-labeled IgE)
h
AJZll AEl8 AE19 AE20 AE21 AE23 AE33 AE34 ‘4E35 AE36 AE38
with a 200 ng of ‘251-labeled IgE mAb 6-16E was incubated varying concentrations of anti-IgE mAb in a volume of 100 ~1. The mixture was added to 1~10~ mast cells (MC/91 in 100 yl and incubated for 90 min before washing. In the absence of inhibitor 30-46 ng of labeled IgE was bound. h Final concentration in the mixture. ’ Not tested.
Concentration
of anti-IgE
500
5
0.5
24 46 6
NT h 35 NT NT NT 69 78 28 97 72 28
NT NT NT NT NT II 0 NT 0 I6 NT
I 30 94 88 38 99 99 40
to B lymof binding
mAb (~g/mll
a The B lymphoma line WEHI (2X 10” cells) was used. The procedure is otherwise the same as that specified in footnote a, Table 3. In the absence of inhibitor 32-46 ng of labeled IgE (6-16E) was bound. b Not tested.
3.4. Inhibition
of binding of IgE to B lymphoma
cells labeled ligand.) Three of the anti-IgE mAbs (AE23, AE33, AE35) were intermediate with respect to inhibitory capacity. The relationship between inhibitory capacity and direct binding to IgE on mast cells (Table 2) will be considered in the Discussion.
Table 5 Degranulation Anti-IgE None ’ NMIgG ’ AEll AE18 AE34 AE36 AE38
mAb
of IgE-sensitized
rat basophils % serotonin 7.7 7.7 6.0 7.0 7.1 7.0 7.0
* * + + f * *
0.2 0.4 0.1 0.8 0.3 0.3 0.3
by anti-IgE
release
b
Four of the anti-IgE mAbs (AE23, AE33, AE35, AE36) strongly inhibited the binding of labeled IgE to B lymphoma cells (Table 4). Five of the mAbs were intermediate in their inhibitory capacity and two (AE19 and AE20) showed little
mAb a Anti-IgE AE23 AE33 AE35 AE21 AE19 AE20
mAb
% serotonin 7.7 29 21 6.6 14 19
release
* 0.6 i3 +2 i_ 0.1 +1 *2
a Rat basophilic leukocytes (RBL-2H3 line), intrinsically labeled with [“Hlserotonin (materials and methods section), were exposed to IgE mAb 6-16E (5 Kg/ml) for 1 h, washed and treated with 200 ~1 of the anti-IgE mAb specified (10 pg/ml) for 90 min at 37” c. b The maximum release of tritium, taken as lOO%, was determined by treatment of control cells with 1% Triton X-100. Experiments were in duplicate. ’ Treated with medium only. ’ Nonspecific mouse IgG.
206
S. Haba. A. Nisonoff /Journal
of Immunological Methods 183 (1995) 199-209
or no inhibitory capacity. The relationship between inhibitory capacity and direct binding to IgE attached to B lymphoma cells (Table 2) will be considered in the discussion section. 3.7. Degranulation of IgE-sensitized rat basophilic Leukocytes and induction of local anaphylaxis by anti-IgE mAbs Four of the 11 anti-IgE mAbs tested (AE19, AE20, AE33 and AE35) caused an extent of degranulation that was significantly greater than that of controls, when exposed to basophilic leukocytes sensitized with IgE (Table 5). Six of 11 mAbs induced immediate local anaphylaxis when inoculated into footpads of CAF, mice (Table 61, whereas the other five mAbs gave negative results. F(ab’), fragments of three of these mAbs were also tested. In each case the results corresponded with those observed with the parent, undigested molecule (two positive, one negative). A summary of properties of the various antiIgE mAbs is shown in Table 7.
4. Discussion We describe here a convenient method for the production of syngeneic anti-IgE mAbs. Its basis is to start the immunization of mice before the age of 2 weeks, using an IgE mAb in saline solution as the immunogen, and to inject this material repeatedly over a period of 4-5 months. This procedure resulted in anti-IgE titers of 500-1300 pg/ml, values high enough to facilitate the production of numerous anti-IgE mAbs. The mean titer is considerably higher than that obtainable by inoculation of a KLH-IgE conjugate into adult mice (Haba and Nisonoff, 1987a). We isolated 26 hybridomas producing anti-IgE mAbs. Seven were idiotype-specific and were not investigated further. Nearly half of our mAbs reacted with Fab fragments of IgE (presumably with the CHl region). This finding contrasts with studies on xenogeneic mAbs against mouse (Baniyash and Eshhar, 1984; Keegan et al., 1991) or human IgE (Hook et al., 1991; Prado et al., 19911, in which the mAbs were predominantly
Fc-directed. We selected ten anti-IgE mAbs for more detailed investigation, together with one from a previous study. Each of these mAbs was shown to be specific for isotypic determinants of IgE and non-reactive with other classes of Ig (Table 1). Their affinities (K,) for IgE ranged from 3.6 X 10’ to 1.1 x 10”’ M-‘; the mean value was about 100 times greater than that obtained with adult recipients, using KLH-IgE in CFA as the immunogen (Haba and Nisonoff, 1987b). We are uncertain as to whether the high affinity resulted from the early immunization of the mice or from the use of IgE in saline, rather than KLH-IgE in CFA. (Adult mice respond very poorly to KLH-IgE in saline.) It may also be relevant that the anti-IgE mAbs obtained by this method are predominantly of the IgGl isotype (18 of 19 clones). IgG2a and IgG2b were the predominant isotypes produced in response to KLH-IgE in CFA (Haba and Nisonoff, 1987b). Such a distinctive isotype distribution may be attributable to the different kinds and/or amounts of lymphokines delivered by helper T cells specific for IgE or for KLH, respectively; this might also influence the maturation of affinity. As noted above, a substantial fraction of the mAbs reacted with the Fab region of IgE. One mAb, AE21, was found to react with F(ab’jz fragments, but not with Fab, of IgE. As the F(ab’), fragment of IgE appears to contain complete CH2 domains (Haba and Nisonoff, 1991), this mAb is probably directed towards CH2. We examined the reactivity of the anti-IgE mAbs with IgE molecules bound to FcgRI or FceRII receptors and observed differential reactivities of the various syngeneic anti-IgE mAbs. The anti-IgE mAbs could be divided into several groups according to their direct binding activity or their capacity to inhibit the binding of IgE to Fee receptors. (Other investigators have shown that human (Helm et al., 1988; Vercelli et al., 1989) and mouse IgE (Nissim et al., 1991; Nissim et al., 1993; Weetall et al., 1990) interact with FcFRI and FcERII through distinct regions of the Fc portion of the IgE molecule.) First, AE21, which appears to be specific for the CH2 domain of IgE, recognizes FcsRI- and
S. Haha. A. Nisonoff /Journal Table 6 Induction Anti-IgE
of local anaphylaxis mAb
ng anti-IgE
100
10
_
_
_
_
NT’ NT NT _
_
NT NT NT _
+ +
+ +
NT + +
+ +
NT -
+
+
+
_
_
+ + + + + -
+ + + + +
+ + + + + _
_
_
+ + _
_
+ _
_
_c _ _ _ _ _
_ _
-
1
” CAF, mice, 3-h-months old, were inoculated in a hindfootpad with 20 ~1 of anti-IgE mAb or NMIgG. The other hind footpad received only diluent (Methods). Control mice received NMIgG in the amounts specified. The mice were immediately inoculated i.v. with 200 ~1 of 0.2% Evans Blue dye in saline. Paws were inspected after 15 min and the mice were killed. ’ Nonspecific mouse IgG. ’ A - or + indicates a negative or a positive (blueing) result, respectively. ’ Not tested.
FcERII-bound IgE but does not inhibit the binding of IgE to either of these receptors. This is obviously consistent with the view that AE21 rec-
Table 7 Summary
of properties
of anti-IgE
Binding sJgE ’
IgE bound to FcsRI ’
IgE bound to FccRII ’
F(ab’1, IgE d
AJZll, AE18, AE34. AE38 AE36 AE23, AE33, AE35 AE21 AJZ19,AE20
+
-
+
_
+ +
_ +
+/+
_
+
+ +
+ +
+ +
” ’ ’ ’
199-209
207
mAbs
Anti-IgE mAb
+
(1995)
ognizes epitopes that are not involved in the binding to either receptor. mAbs in a second group - AEl 1, AE18, AE34 and AE38 - do not recognize IgE bound to FceRI but do react with FceRII-bound IgE. They also inhibit the binding of IgE to FcERI but do not inhibit binding to FceRII. This indicates that these four mAbs interact with epitopes that are directly involved (or sterically blocked) in the binding to FcFRI and that remain exposed when IgE is bound to FcsRII. AE36 represents a third type of specificity. It does not interact with FcERI-bound IgE and interacts very poorly with FcERII-bound IgE, but it can inhibit the binding of IgE to both FcFRI and FceRII. The latter observation suggests that the epitopes involved in binding to FcFRI and FceRII are in close enough proximity so that a single mAb can interfere with the binding of IgE to either receptor. (That nonidentical regions of IgE bind to the two receptors is shown by the patterns of binding of other anti-IgE mAbs.) mAbs AE19 and AE20, which are directed against the Fab of IgE, have, as expected, no competitive effects on the binding of IgE to FcsRI or FceRII. Such differential binding and inhibitory activities of anti-IgE mAbs are consistent with previously reported observations, made with xenogeneic anti-mouse or anti-human IgE, that the binding sites of IgE that interact with FcPRI or FcERII are distinct but are located in close prox-
mAbs ’
mAb inoculated 1000
10000 NMIgG h AEll AElf! AE34 AE36 AE38 AE19 AE20 AE2 I AJS23 AE33 AE35 AE19 Ffab’), AE33 F(ab’1, AE38 Flab’),
by anti-IgE
of Immunological Methods 153
to
JgE present on the surface of a B cell hybridoma. Present on a murine mast cell line. Present on a murine B Jymphoma cell line. Bound to a plastic surface.
of
Fab of JgE d
Inhibition of IgE binding to
In vivo anaphylaxis
FcERJ
FcPRJI
+
_
_
_ _
+ +
+ +
_
_
_ _
_
+ +
+
+
208
S. Haba, A. Nisonoff / Journal of Immunological Methods 183 (1995) 199-209
imity to one another (Baniyash and Eshhar, 1984; Baniyash et al., 1988; Davis et al., 1993; Hook et al., 1991; Rings et al., 1986). Another type of specificity is associated with mAbs AE23, AE33 and AE35. Each of these mAbs were bound to IgE attached to either FcsRI or FcsRII. However, in contrast to AE19, AE20 and AE21 (which also interacted with IgE bound to either receptor) AE23, AE33 and AE3.5 were able to inhibit the binding of IgE to each receptor. A possible explanation is that the determinants on IgE with which these mAbs interact are repeated on the IgE molecule in such a way that at least one determinant remains exposed when the IgE bound to a receptor. This interpretation requires the additional assumption that the affinities of the mAbs are such that they can prevent binding when added to the IgE before exposure to the cells but displace bound IgE very slowly or not at all. There is an excellent correlation (Tables 6 and 71 between the capacity of each of the mAbs tested to interact with IgE bound to FceRI and their ability to cause anaphylactic reactions in vivo (in the paw of the mouse). Other investigators have used xenogeneic antiIgE antibodies to modulate IgE antibody production in vivo (Bozelka et al., 1982,1985; Davis et al., 1993; Dessein et al., 1981). A possible drawback for the use of xenogeneic Ig for suppression of IgE synthesis is its immunogenicity. Our studies have focused on the suppression of IgE production by syngeneic monoclonal or polyclonal anti-IgE antibodies (Haba and Nisonoff, 1990,1994a,1994b). From a practical standpoint, the fine specificity of these anti-IgE antibodies may be of great importance for the avoidance of anaphylactic reactions during suppression. The anti-IgE mAbs described here may be useful for the establishment of a mouse model for effective suppression of IgE in humans. In this context, Davis et al. (1991) have described mouse mAbs to human IgE that are specific for membraneanchoring segments of IgE heavy chains present on IgE-producing B cells. They did not react with IgE in solution or with IgE bound to FcERI or FcsRII. Antibodies with appropriate characteristics may prove to be therapeutically useful.
Acknowledgements
This work was supported by Grant IM-607C from the American Cancer Society. We are grateful to Ms. T. Haba for technical assistance.
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