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Functional (ADCC) study of 54 IgG MAb anti-RhD
TCB 1996
6:459-463
Functional [ADCC] study of 54 IgG MAb anti-RhD Y. BROSSARD*, C1. D A N G U * , C. PATEREAU**, D. GOOSSENS** * Centre d'H~mobiologie P~rinatale, Paris, France ** Institut National de la Transfusion Sanguine, Paris, France
Introduction Rh i m m u n e suppression is a c o m p l e x Fc-mediated process involving IgG-anti-D a n t i b o d y and i m m u n e cells bearing Fc receptors, m a i n l y B cells [1]. It begins w i t h the Fc m e d i a t e d clearance of Rh * red b l o o d cells (RBCs) b y s p l e e n m a c r o p h a g e s and experimental evidence suggests a strong positive association b e t w e e n the i m m u n e clearance of RBCs and i m m u n e suppression against Rh D antigen. While Rh p l a s m a i m m u n o g l o b u l i n s regularly induce a rapid in vivo clearance of Rh m RBCs, this is not the case with all IgG anti-D mAbs. Only anti-D m A b s properly selected with relevant cellular (hemolytic) assays m a y eventually be strongly h e m o l y t i c in vivo a n d m a y express an i m m u n o - s u p p r e s s i v e capacity [2].
Cellular assays usually w o r k in a rather artificial context and false positive as well as false negative results m a y be observed for the prediction of the in vivo h e m o l y t i c p o t e n c y of I g G anti-D antibodies [3]. In the present study we focused on an ADCC syst e m taking into a c c o u n t b o t h externally lysed and phagocytosed RBCs, and we comparatively tested 54 IgG anti-D m A b s w i t h several populations of effector cells against polyclonal I g G anti-D antibodies f r o m h u m a n Rh i m m u n o g l o b u l i n .
Methods A D C C assays T h e y a r e s u m m a r i z e d o n t a b l e 1.
Table 1
Technical parameters of the four ADCC assays Effector cells
Incubation
/RBC ratio
time
Monocytes-Kcells Macrophages
~- 1
PBMCminus monocytes
Kcells
Macrophage
Adherent monocytes
Monocyte
PBMC
ADCC
Cells
PBMC
PBMC
Lymphocyte
Fc3,R
RBCs
Human serum
18 h
enzyme treated
yes
I (II ?) III
~ 0.5
18 h
enzyme treated
yes
III
Macrophages
10
18 h
normal
yes
I (II ?) III
Monocytes
~1
3h
normal pre-sensitized
no
I
Effector cells
involved
Correspondence to: Y. Brossard, Centre d'Hdmobiologie Pdrinatale,
53, bd Diderot, 75012 Paris, France. 459
460
Y. BROSSARD E T AL.
The assay using peripheral blood monuclear cells (ADCC-PBMC) is described in the annex. The other assays differed as follows: - ADCC-1ymphocvte (ADCC-Ly): Two ml of PBMcell (107/ml) suspension were delivered onto a thin layer of neutral AB serum covering the bottom of a glass Petri dish (diameter 7 cm). After an incubation time of 0.5 hours (at 37°C - 5% CO2), n o n a d h e r e n t cells were retrieved then w a s h e d with PBS-Albumin and finally adjusted to 6 x 106/ml. The assay was then carried out like the ADCC-PBMC. - ADCC-Macrophages (ADCC-macro): The above Petri dishes were washed three times with PBSAlbumin to eliminate residual non a d h e r e n t cells. Adherent m o n o c y t e s were retrieved after 10 m n incubation with 2.5 ml of a cell dissociation non-enzymatic solution (SIGMA C 5914). After three washes with PBS-Albumin, the monocytes were adjustod to 10 x 106/ml. Normal R1, R z RBCs w e r e used in the assay (50/~1 at 2 x 106/ml) together with 100/d of monocyte syspension (effector/target ratio = 10). - ADCC-Monocytes (ADCC-Mono): 2 x 106 normal Rzr RBCs w e r e sensitized (1 hour - 37°C) with 100 td of undiluted antibody supernatant. After three washes, these were adjusted at one ml and 50 ~1 w e r e incubated with 5 0 / d of PBMC (107/ml) during 3 hours, in the absence of neutral AB serum.
Anti-D antibodies 54 IgG anti-D antibodies w e r e selected among the azide-free antibody supernatants (see footnote)*. Unless indicated they w e r e used undiluted. An Rh immunoglobnlin (WinRho-S.D, Winnipeg, Canada) diluted at 4/~g of polyclonal IgG anti-D per ml served as an internal standard.
Design of the study: - The 54 IgG mAbs were tested in parallel in three ADCC assays (ADCC-PBMC, ADCC-Ly, ADCCMacro). At least three experiments w e r e performed, each with a different donor of effector cells. A specific activity at or above 40% was required with the polyclonal anti-D. These three ADCC assays w e r e selected for the screening * 31 to 33, 36, 45 to 49, 63 to 68, 70 to79, 81 to 83, 88, 89, 93 to 97, 101, 102, 104 to 106, 108, 114, 117 to 124, 130, 131, 181.
step of the study because they w e r e thought to be rather c o m p l e m e n t a r y at identifying the most powerful antibodies. As described above, normal RBCs or RBCs treated with minute amounts of papain were used, and neutral AB serum served as a source of competitive monomerle IgG in all three assays. The most active antibodies in ADCC-Macro w e r e further tested with this assay b y decreasing the effector/target ratio from 10 to 3, then to 1. This was done to better define the relative p o t e n c y of the antibodies in this assay. T h r e e IgG mAbs already tested in vivo (n ° 104, n ° 105, n ° 93) were compared, in two parallel experiments, in ADCC-Ly, ADCC-Mono, and ADCC-Macro. After measuring their concentration on Autoanalyser ®, the antibodies were diluted at 1/zg/ml. The RBCs used in the ADCCMono were pre-sensitized with undiluted supernatants. This experiment was carried out to clarify the preferential FcR c o m m i t m e n t of the monoclonal antibodies and to test the predictive value of the three assays.
Results 44 out of 54 antibodies w e r e active in ADCC-PBMC but only 9 and 16 of these antibodies were also hemolytic in ADCC-Ly and ADCC-Macro respectively {Table 2). Three antibodies In°s 70, 105, 123) that w e r e inactive in ADCC-PBMC and ADCC-Ly displayed a significant hemolytic activity in ADCCMacro. Some antibodies were at least as hemolytic as the polyclonal IgG anti-D standard in one assay (n °s 66, 67, 73, 81, 82, 89), in two assays (n ° 88), and in the three assays (n ° 104). The 9 antibodies active in ADCC-Ly w e r e also highly hemolytic in ADCC-PBMC {mean specific hemolysis: 72%) suggesting a partial identity between the two assays. Indeed, the assays differed only by m o n o c y t e s that were present at a low effector/target ratio (~ 0.5) with the ADCC-PBMC. Among the 19 antibodies reactive in ADCC-Macro, 5 w e r e also hemolytic in ADCC-Ly [3/3 w h e n the specific hemolysis in ADCC-Ly was > 30%, and only 2/6 w h e n it was < 30%). No clear correlation was observed b e t w e e n the results of the ADCCMacro and those of the ADCC-PBMC, w h e n considering the seven antibodies most reactive in ADCC-Macro (specific hemolysis > 40%) that were
FUNCTIONAL (ADCC) STUDY OF 54 IgG MAB ANTI-RHD
461
Table 2 Activity of t h e 54 IgG m A b anti-D in three A D C C a s s a y s M o n o c l o n a l antibodies are identified b y their code n u m b e r ; Ig Rh = Rh i m m u n o g l o b u l i n diluted a 4/~g IgG anti-D/ml. O n l y antibodies active at or above 15 % specific h e m o l y s i s are indicated. E/RBC = Effector/target ceils ratio.
Specific h e m o l y s i s (%)
ADCC-PBMC (mean of ~> 3 experiments}
15%
20%
32
rxm145 65
[]
94
[] 95, 114, 121, 131,
30% 48 36 6875
40% 49 30 7693
50%
60%
70%
47, 64
72 124 [ ]
97
80%
31, 33 ~46, 71
77
90%
100% 104
67
vaG31001,73, Ig Rh
102 120 9 6 130 181
E/RBC: = 1
101, 118 122
ADCC-LY (mean of ~> 3 experiments)
31, 45 71, 97
46, 67
33, 73
33,71 72, 77 93
31,68 102
73
Ig Rh
104
70 new
104 ]Ig Rh
E/RBC: = 0.5 ADCC"Macro" m e a n of 3 experiments}
[]
[]
[] []
[]
E/RBC: 10 IgG3: ~ / ~ ; T h e other antibodies w e r e typed as I g G n in t h e W o r k s h o p
ADCC"Macro" (mean of 2 experiments} E/RBC: 1
104
[]
[]
[]
{The following antibodies w e r e tested at this E/RBC ratio; n °s 66, 70, 78, 81, 82, 88, 104, 105, Ig Rh)
inactive in ADCC-Ly; with the exception of antibody n ° 88, t h e y w e r e either unreactive or m o d e r a t e l y reactive in ADCC-PBMC (mean specific hemolysis: 30%). This could be explained b y a major difference in the macrophage/RBC ratio b e t w e e n the two assays; w h e n this ratio was lowered from 10 to 1 in the ADCC-Macro assay, the relative p o t e n c y of the seven antibodies was highlighted (table II-bottom) and the results were roughly equivalent to those observed in ADCC-PBMC. W h e n considering the subclass repartition of the most highly reactive antibodies in ADCC-Ly and ADCC-Macro, an excess of IgG 1 could be noted in the former, and of IgG 3 in the latter. The ADCC results presented on table 3 confirmed the functional dichotomy of the two monoclonal anti-
Table 3 Comparative ADCC s t u d y of the 3 IgG m A b s previously tested in uiuo (two experiments) S p e c i f i c h e m o l y s i s : O: < 15%; +: 15-40%; + +: 40-70%; + + + : > 70% ADCC
Ig Rh
n ° 104
N ° 105
N ° 93
Mono
+++
0/+
++ +
+
Ly
++
++ +
0/+
0/+ +
Macro
+ +/+ + +
+ +
+/+ + +
0
bodies shown to be hemolytic in vivo (n ° 104 an IgG z, acting via Fc3,R III bearing cells and n ° 105, an IgG a acting via Fc3,R I bearing cells) [2]. Both
462
Y. BROSSARD E T A L .
antibodies were highly reactive in ADCC-Macro, while n ° 93, an antibody poorly hemolytic in vivo [4], was inactive.
D i s c u s s i o n
In this study, the ADCC hemolytic score of the 54 IgG mAb anti-D varied widely and few antibodies displayed a hemolytic potency at or above that of the polyclonal. When considering the ADCC results of three IgG mAbs which have been tested in vivo, t h e ADCC-macrophage assay appeared to segregate rather well the two hemolytic antibodies from the poorly hemolytic one. Yet, this last antibody could be classified in an intermediate position between the ADCCinactive and the highly ADCC-reactive antibodies. Among the latter, our results are in accordance with the previously described dichotomy between 2gGs mAb acting preferentially through Fc3,R I receptors and IgG 1 acting mainly through Fc3,R 112 receptors. In this respect, two prototype antibodies emerged in the study, n ° 104 and n ° 88. The former, an IgG 1, was by far the most reactive antibody in ADCC-Ly but it did not induce a significant hemolysis with monocytes in short-term cultures. However, when using the same monocytes purified by glass adherence, then cultured for 18 h in the presence of norreal RBCs and human serum, the antibody displayed a high hemolytic score. This might be interpreted as the consequence of the appearance of FcyR II2 on the monocyte membrane [5]. The second antibody, an IgGa, was shown to be the most hemolytic antibody in ADCC-Macro when the effector/target cell ratio was decreased; this antibody probably acted mainly through Fc-~R I, being quite inactive in ADCC-Ly but highly hemolytic (> 70%) in ADCC-Mono.
AB human neutral serum RPMI medium: RPMI 1640 (G2BCO) with 1% (w/v} human albumin and 1% {v/v), antibioticantimycotic GIBCO Ref. 1814. PBS-Albumin: pH7 with 1% (w/v) human albumin NH4 C1 Iysis solution: NH4 CI: 4.16 g Nail COa: 0.42 g Na 2 EDTA, 2H20:21.6 mg Distilled H20:500 ml - Phosphate citrate buffer with sodium perborate (capsules): SIGMA Ref. P 4922 - p h e n y l e n e d i a m i n e dihydrochloride 10 mg (tablets) SIGMA Ref. P 8287.20 mg in 25 ml of phosphatecitrate Buffer H204, 2 N solution. -
MATERIAL: 96 U wells microtiter plates CO 2 incubator Orbital shaker Elisa reader for microtiter plates (filter: 592 mm - reference filter at 630 mn). Calibration control: DRI-DYE (awareness technology Inc. Palm city. F1. USA) CELL SUSPENSIONS: -
-
Annex: ADCC - P e r i p h e r a l b l o o d Cells (ADCC - PBMC). P r o c e d u r e
mononuclear
REAGENTS: - Human red blood cells: O Rz/r (blood collected and stored on CPD or ACD) - Fresh peripheral blood mononuclear cells (PBMC) isolated on Ficoll-Hypaque from whole blood collected on K3 EDTA Papaine-cgstein solution: water-soluble papain MERCK ref.: 7 144 diluted at 1% (w/v) in Hendry buffer pH:7.2
P B M C : Cells collected at the ficoll-hypaque
interface are washed twice with PBS-Alb. Residual RBC are lysed with NH4 C1 solution during 10 mn at 4°C. PBMC are then washed twice with PBS-Alb; finally, cells are adjusted to 1.107/ml in RPMI medium. R B C : Papainization: saline-washed RBC are incubated with five volumes of a 1/1000 PBS dilution of the 1% papain-cystein solution (final concentration 1/100 000), during 15 mn at 37°C. Red blood cells are then washed and adjusted to 2.10 s cells/ml in RPMI medium.
ADCC: Deliver the reagents in the U wells in the following order: • Reaction wells (duplicates or triplicates): 10/zpl of the antibody solution 10/d of neutral AB serum
F U N C T I O N A L (ADCC) S T U D Y O F 54 I g G M A B A N T I - R H D
50/~1 of P B M C s u s p e n s i o n 50/~1 of RBC s u s p e n s i o n • C o n t r o l s wells (triplicates): Control A : O m i t the a n t i b o d y solution C o n t r o l B: O m i t the RBC s u s p e n s i o n • Reference antibody: T h e r e f e r e n c e a n t i b o d y (Rh i m m u n o g l o b u l i n a d j u s t e d a 4 ~g I g G anti-D p e r ml) is p r o c e s s e d as the a n t i b o d y s a m p l e u n d e r study. I n c u b a t e during 18-24 h o u r s at 3 7 ° C in air enric h e d at 5% CO 2 w a s h the wells 3 t i m e s w i t h R P M I m e d i u m . Lyse t h e residual RBCs w i t h 150 #1 N H 4 C1 at 4 ° C d u r i n g 10 m n o n a n orbital shaker. Centrifuge. T r a n s f e r 100/xl of s u p e r n a t a n t into a well of a U - b o t o m m e d titer m i c r o p l a t e . A d d O P D solution (100 #1 p e r well) a n d i n c u b a t e at 3 7 ° C d u r i n g 20 m n . Stop t h e r e a c t i o n w i t h 50 ~1 H2SO 4 2N. R e a d the a b s o r b a n c e at 492 m n . -
-
-
-
-
463
E X P R E S S I O N O F T H E RESULTS: T h e m e a n O D of e a c h s a m p l e is expressed in t e r m s of specific h e m o l y s i s (%) in the f o l l o w i n g w a y : Specific h e m o l y s i s (%) = OD Control A - OD sample
X 100
OD control A - control B
R
e
f
e
r
e
n
c
e
s
[1] Heyman B. (1990) The immune complex: possible ways of regulating the antibody response. Imrnunol. today, 11,310-313. [2] Kumpei B.M., Goodrick M.J., Pamphilon D.H., Fraser I.D., Poole G.D., Morse C., Standen G.R, Chapman G.E., Thomas D.P., Anstee D.J. (1995) Human monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D red Blood Cells and suppression of Rh D immunization in Rh D negative volunteers. Blood, 86, 1071-1079. [3] Zupafiska B. (1994) Cellular immunosssays and their use for predicting the clinical significance of antibodies in Immunobiology of Transfusion Medecine (G Garatty ed.) 1 vol. (M. Dekker N.Y. USA): 465-491. [4] Brossard Y. et aL (1990) (unpublished observations). [5] Ravetch J.V, Kinet J.P. (1991) Fc receptors. Ann. Rev. Immunol., 9, 457-492.
6:459-463
Functional [ADCC] study of 54 IgG MAb anti-RhD Y. BROSSARD*, C1. D A N G U * , C. PATEREAU**, D. GOOSSENS** * Centre d'H~mobiologie P~rinatale, Paris, France ** Institut National de la Transfusion Sanguine, Paris, France
Introduction Rh i m m u n e suppression is a c o m p l e x Fc-mediated process involving IgG-anti-D a n t i b o d y and i m m u n e cells bearing Fc receptors, m a i n l y B cells [1]. It begins w i t h the Fc m e d i a t e d clearance of Rh * red b l o o d cells (RBCs) b y s p l e e n m a c r o p h a g e s and experimental evidence suggests a strong positive association b e t w e e n the i m m u n e clearance of RBCs and i m m u n e suppression against Rh D antigen. While Rh p l a s m a i m m u n o g l o b u l i n s regularly induce a rapid in vivo clearance of Rh m RBCs, this is not the case with all IgG anti-D mAbs. Only anti-D m A b s properly selected with relevant cellular (hemolytic) assays m a y eventually be strongly h e m o l y t i c in vivo a n d m a y express an i m m u n o - s u p p r e s s i v e capacity [2].
Cellular assays usually w o r k in a rather artificial context and false positive as well as false negative results m a y be observed for the prediction of the in vivo h e m o l y t i c p o t e n c y of I g G anti-D antibodies [3]. In the present study we focused on an ADCC syst e m taking into a c c o u n t b o t h externally lysed and phagocytosed RBCs, and we comparatively tested 54 IgG anti-D m A b s w i t h several populations of effector cells against polyclonal I g G anti-D antibodies f r o m h u m a n Rh i m m u n o g l o b u l i n .
Methods A D C C assays T h e y a r e s u m m a r i z e d o n t a b l e 1.
Table 1
Technical parameters of the four ADCC assays Effector cells
Incubation
/RBC ratio
time
Monocytes-Kcells Macrophages
~- 1
PBMCminus monocytes
Kcells
Macrophage
Adherent monocytes
Monocyte
PBMC
ADCC
Cells
PBMC
PBMC
Lymphocyte
Fc3,R
RBCs
Human serum
18 h
enzyme treated
yes
I (II ?) III
~ 0.5
18 h
enzyme treated
yes
III
Macrophages
10
18 h
normal
yes
I (II ?) III
Monocytes
~1
3h
normal pre-sensitized
no
I
Effector cells
involved
Correspondence to: Y. Brossard, Centre d'Hdmobiologie Pdrinatale,
53, bd Diderot, 75012 Paris, France. 459
460
Y. BROSSARD E T AL.
The assay using peripheral blood monuclear cells (ADCC-PBMC) is described in the annex. The other assays differed as follows: - ADCC-1ymphocvte (ADCC-Ly): Two ml of PBMcell (107/ml) suspension were delivered onto a thin layer of neutral AB serum covering the bottom of a glass Petri dish (diameter 7 cm). After an incubation time of 0.5 hours (at 37°C - 5% CO2), n o n a d h e r e n t cells were retrieved then w a s h e d with PBS-Albumin and finally adjusted to 6 x 106/ml. The assay was then carried out like the ADCC-PBMC. - ADCC-Macrophages (ADCC-macro): The above Petri dishes were washed three times with PBSAlbumin to eliminate residual non a d h e r e n t cells. Adherent m o n o c y t e s were retrieved after 10 m n incubation with 2.5 ml of a cell dissociation non-enzymatic solution (SIGMA C 5914). After three washes with PBS-Albumin, the monocytes were adjustod to 10 x 106/ml. Normal R1, R z RBCs w e r e used in the assay (50/~1 at 2 x 106/ml) together with 100/d of monocyte syspension (effector/target ratio = 10). - ADCC-Monocytes (ADCC-Mono): 2 x 106 normal Rzr RBCs w e r e sensitized (1 hour - 37°C) with 100 td of undiluted antibody supernatant. After three washes, these were adjusted at one ml and 50 ~1 w e r e incubated with 5 0 / d of PBMC (107/ml) during 3 hours, in the absence of neutral AB serum.
Anti-D antibodies 54 IgG anti-D antibodies w e r e selected among the azide-free antibody supernatants (see footnote)*. Unless indicated they w e r e used undiluted. An Rh immunoglobnlin (WinRho-S.D, Winnipeg, Canada) diluted at 4/~g of polyclonal IgG anti-D per ml served as an internal standard.
Design of the study: - The 54 IgG mAbs were tested in parallel in three ADCC assays (ADCC-PBMC, ADCC-Ly, ADCCMacro). At least three experiments w e r e performed, each with a different donor of effector cells. A specific activity at or above 40% was required with the polyclonal anti-D. These three ADCC assays w e r e selected for the screening * 31 to 33, 36, 45 to 49, 63 to 68, 70 to79, 81 to 83, 88, 89, 93 to 97, 101, 102, 104 to 106, 108, 114, 117 to 124, 130, 131, 181.
step of the study because they w e r e thought to be rather c o m p l e m e n t a r y at identifying the most powerful antibodies. As described above, normal RBCs or RBCs treated with minute amounts of papain were used, and neutral AB serum served as a source of competitive monomerle IgG in all three assays. The most active antibodies in ADCC-Macro w e r e further tested with this assay b y decreasing the effector/target ratio from 10 to 3, then to 1. This was done to better define the relative p o t e n c y of the antibodies in this assay. T h r e e IgG mAbs already tested in vivo (n ° 104, n ° 105, n ° 93) were compared, in two parallel experiments, in ADCC-Ly, ADCC-Mono, and ADCC-Macro. After measuring their concentration on Autoanalyser ®, the antibodies were diluted at 1/zg/ml. The RBCs used in the ADCCMono were pre-sensitized with undiluted supernatants. This experiment was carried out to clarify the preferential FcR c o m m i t m e n t of the monoclonal antibodies and to test the predictive value of the three assays.
Results 44 out of 54 antibodies w e r e active in ADCC-PBMC but only 9 and 16 of these antibodies were also hemolytic in ADCC-Ly and ADCC-Macro respectively {Table 2). Three antibodies In°s 70, 105, 123) that w e r e inactive in ADCC-PBMC and ADCC-Ly displayed a significant hemolytic activity in ADCCMacro. Some antibodies were at least as hemolytic as the polyclonal IgG anti-D standard in one assay (n °s 66, 67, 73, 81, 82, 89), in two assays (n ° 88), and in the three assays (n ° 104). The 9 antibodies active in ADCC-Ly w e r e also highly hemolytic in ADCC-PBMC {mean specific hemolysis: 72%) suggesting a partial identity between the two assays. Indeed, the assays differed only by m o n o c y t e s that were present at a low effector/target ratio (~ 0.5) with the ADCC-PBMC. Among the 19 antibodies reactive in ADCC-Macro, 5 w e r e also hemolytic in ADCC-Ly [3/3 w h e n the specific hemolysis in ADCC-Ly was > 30%, and only 2/6 w h e n it was < 30%). No clear correlation was observed b e t w e e n the results of the ADCCMacro and those of the ADCC-PBMC, w h e n considering the seven antibodies most reactive in ADCC-Macro (specific hemolysis > 40%) that were
FUNCTIONAL (ADCC) STUDY OF 54 IgG MAB ANTI-RHD
461
Table 2 Activity of t h e 54 IgG m A b anti-D in three A D C C a s s a y s M o n o c l o n a l antibodies are identified b y their code n u m b e r ; Ig Rh = Rh i m m u n o g l o b u l i n diluted a 4/~g IgG anti-D/ml. O n l y antibodies active at or above 15 % specific h e m o l y s i s are indicated. E/RBC = Effector/target ceils ratio.
Specific h e m o l y s i s (%)
ADCC-PBMC (mean of ~> 3 experiments}
15%
20%
32
rxm145 65
[]
94
[] 95, 114, 121, 131,
30% 48 36 6875
40% 49 30 7693
50%
60%
70%
47, 64
72 124 [ ]
97
80%
31, 33 ~46, 71
77
90%
100% 104
67
vaG31001,73, Ig Rh
102 120 9 6 130 181
E/RBC: = 1
101, 118 122
ADCC-LY (mean of ~> 3 experiments)
31, 45 71, 97
46, 67
33, 73
33,71 72, 77 93
31,68 102
73
Ig Rh
104
70 new
104 ]Ig Rh
E/RBC: = 0.5 ADCC"Macro" m e a n of 3 experiments}
[]
[]
[] []
[]
E/RBC: 10 IgG3: ~ / ~ ; T h e other antibodies w e r e typed as I g G n in t h e W o r k s h o p
ADCC"Macro" (mean of 2 experiments} E/RBC: 1
104
[]
[]
[]
{The following antibodies w e r e tested at this E/RBC ratio; n °s 66, 70, 78, 81, 82, 88, 104, 105, Ig Rh)
inactive in ADCC-Ly; with the exception of antibody n ° 88, t h e y w e r e either unreactive or m o d e r a t e l y reactive in ADCC-PBMC (mean specific hemolysis: 30%). This could be explained b y a major difference in the macrophage/RBC ratio b e t w e e n the two assays; w h e n this ratio was lowered from 10 to 1 in the ADCC-Macro assay, the relative p o t e n c y of the seven antibodies was highlighted (table II-bottom) and the results were roughly equivalent to those observed in ADCC-PBMC. W h e n considering the subclass repartition of the most highly reactive antibodies in ADCC-Ly and ADCC-Macro, an excess of IgG 1 could be noted in the former, and of IgG 3 in the latter. The ADCC results presented on table 3 confirmed the functional dichotomy of the two monoclonal anti-
Table 3 Comparative ADCC s t u d y of the 3 IgG m A b s previously tested in uiuo (two experiments) S p e c i f i c h e m o l y s i s : O: < 15%; +: 15-40%; + +: 40-70%; + + + : > 70% ADCC
Ig Rh
n ° 104
N ° 105
N ° 93
Mono
+++
0/+
++ +
+
Ly
++
++ +
0/+
0/+ +
Macro
+ +/+ + +
+ +
+/+ + +
0
bodies shown to be hemolytic in vivo (n ° 104 an IgG z, acting via Fc3,R III bearing cells and n ° 105, an IgG a acting via Fc3,R I bearing cells) [2]. Both
462
Y. BROSSARD E T A L .
antibodies were highly reactive in ADCC-Macro, while n ° 93, an antibody poorly hemolytic in vivo [4], was inactive.
D i s c u s s i o n
In this study, the ADCC hemolytic score of the 54 IgG mAb anti-D varied widely and few antibodies displayed a hemolytic potency at or above that of the polyclonal. When considering the ADCC results of three IgG mAbs which have been tested in vivo, t h e ADCC-macrophage assay appeared to segregate rather well the two hemolytic antibodies from the poorly hemolytic one. Yet, this last antibody could be classified in an intermediate position between the ADCCinactive and the highly ADCC-reactive antibodies. Among the latter, our results are in accordance with the previously described dichotomy between 2gGs mAb acting preferentially through Fc3,R I receptors and IgG 1 acting mainly through Fc3,R 112 receptors. In this respect, two prototype antibodies emerged in the study, n ° 104 and n ° 88. The former, an IgG 1, was by far the most reactive antibody in ADCC-Ly but it did not induce a significant hemolysis with monocytes in short-term cultures. However, when using the same monocytes purified by glass adherence, then cultured for 18 h in the presence of norreal RBCs and human serum, the antibody displayed a high hemolytic score. This might be interpreted as the consequence of the appearance of FcyR II2 on the monocyte membrane [5]. The second antibody, an IgGa, was shown to be the most hemolytic antibody in ADCC-Macro when the effector/target cell ratio was decreased; this antibody probably acted mainly through Fc-~R I, being quite inactive in ADCC-Ly but highly hemolytic (> 70%) in ADCC-Mono.
AB human neutral serum RPMI medium: RPMI 1640 (G2BCO) with 1% (w/v} human albumin and 1% {v/v), antibioticantimycotic GIBCO Ref. 1814. PBS-Albumin: pH7 with 1% (w/v) human albumin NH4 C1 Iysis solution: NH4 CI: 4.16 g Nail COa: 0.42 g Na 2 EDTA, 2H20:21.6 mg Distilled H20:500 ml - Phosphate citrate buffer with sodium perborate (capsules): SIGMA Ref. P 4922 - p h e n y l e n e d i a m i n e dihydrochloride 10 mg (tablets) SIGMA Ref. P 8287.20 mg in 25 ml of phosphatecitrate Buffer H204, 2 N solution. -
MATERIAL: 96 U wells microtiter plates CO 2 incubator Orbital shaker Elisa reader for microtiter plates (filter: 592 mm - reference filter at 630 mn). Calibration control: DRI-DYE (awareness technology Inc. Palm city. F1. USA) CELL SUSPENSIONS: -
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Annex: ADCC - P e r i p h e r a l b l o o d Cells (ADCC - PBMC). P r o c e d u r e
mononuclear
REAGENTS: - Human red blood cells: O Rz/r (blood collected and stored on CPD or ACD) - Fresh peripheral blood mononuclear cells (PBMC) isolated on Ficoll-Hypaque from whole blood collected on K3 EDTA Papaine-cgstein solution: water-soluble papain MERCK ref.: 7 144 diluted at 1% (w/v) in Hendry buffer pH:7.2
P B M C : Cells collected at the ficoll-hypaque
interface are washed twice with PBS-Alb. Residual RBC are lysed with NH4 C1 solution during 10 mn at 4°C. PBMC are then washed twice with PBS-Alb; finally, cells are adjusted to 1.107/ml in RPMI medium. R B C : Papainization: saline-washed RBC are incubated with five volumes of a 1/1000 PBS dilution of the 1% papain-cystein solution (final concentration 1/100 000), during 15 mn at 37°C. Red blood cells are then washed and adjusted to 2.10 s cells/ml in RPMI medium.
ADCC: Deliver the reagents in the U wells in the following order: • Reaction wells (duplicates or triplicates): 10/zpl of the antibody solution 10/d of neutral AB serum
F U N C T I O N A L (ADCC) S T U D Y O F 54 I g G M A B A N T I - R H D
50/~1 of P B M C s u s p e n s i o n 50/~1 of RBC s u s p e n s i o n • C o n t r o l s wells (triplicates): Control A : O m i t the a n t i b o d y solution C o n t r o l B: O m i t the RBC s u s p e n s i o n • Reference antibody: T h e r e f e r e n c e a n t i b o d y (Rh i m m u n o g l o b u l i n a d j u s t e d a 4 ~g I g G anti-D p e r ml) is p r o c e s s e d as the a n t i b o d y s a m p l e u n d e r study. I n c u b a t e during 18-24 h o u r s at 3 7 ° C in air enric h e d at 5% CO 2 w a s h the wells 3 t i m e s w i t h R P M I m e d i u m . Lyse t h e residual RBCs w i t h 150 #1 N H 4 C1 at 4 ° C d u r i n g 10 m n o n a n orbital shaker. Centrifuge. T r a n s f e r 100/xl of s u p e r n a t a n t into a well of a U - b o t o m m e d titer m i c r o p l a t e . A d d O P D solution (100 #1 p e r well) a n d i n c u b a t e at 3 7 ° C d u r i n g 20 m n . Stop t h e r e a c t i o n w i t h 50 ~1 H2SO 4 2N. R e a d the a b s o r b a n c e at 492 m n . -
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463
E X P R E S S I O N O F T H E RESULTS: T h e m e a n O D of e a c h s a m p l e is expressed in t e r m s of specific h e m o l y s i s (%) in the f o l l o w i n g w a y : Specific h e m o l y s i s (%) = OD Control A - OD sample
X 100
OD control A - control B
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[1] Heyman B. (1990) The immune complex: possible ways of regulating the antibody response. Imrnunol. today, 11,310-313. [2] Kumpei B.M., Goodrick M.J., Pamphilon D.H., Fraser I.D., Poole G.D., Morse C., Standen G.R, Chapman G.E., Thomas D.P., Anstee D.J. (1995) Human monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D red Blood Cells and suppression of Rh D immunization in Rh D negative volunteers. Blood, 86, 1071-1079. [3] Zupafiska B. (1994) Cellular immunosssays and their use for predicting the clinical significance of antibodies in Immunobiology of Transfusion Medecine (G Garatty ed.) 1 vol. (M. Dekker N.Y. USA): 465-491. [4] Brossard Y. et aL (1990) (unpublished observations). [5] Ravetch J.V, Kinet J.P. (1991) Fc receptors. Ann. Rev. Immunol., 9, 457-492.