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Human monoclonal antibodies against blood group antigens
Journal of Immunological Methods, 101 (1987) 193-200
193
Elsevier JIM 04408
Human monoclonal antibodies against blood group antigens Preparation of a series of stable EBV immortalized B clones producing high levels of antibody of different isotypes and specificities Dominique Goossens 1, Fran~oise Champomier 1, Philippe Rouger 2 and Charles Salmon 2 i Centre National de Rkfkreneepour les Groupes Sanguins, CNTS Institut, Paris, and e Universit~ Pierre et Marie Curie, 75571 Paris Cedex 12, France
(Received 24 November 1986, revised received 22 January 1987, accepted 17 March 1987)
The EBV immortalization technique was used to produce stable clones, from B lymphocytes, secreting human monoclonal antibodies to Rh(D), Rh(G), Rh(c), Rh(E), Kell, A and A 1 blood group antigens. These clones were obtained from peripheral blood lymphocytes of hyperimmunized plasmapheresis donors or from spleen lymphocytes of immunized patients. Mean levels of antibody concentration varied between 4 and 50 #g/ml. The antibodies obtained were of IgG~, IgG2, IgM or IgA class. Most of the clones have been stable for growth and antibody production during long periods of continuous culture, extending upto 4 years. Hybridization of two clones was effected with the human lymphoblastoid cell line KR-4 and with the mouse myeloma X63-Ag8.653, but did not result in any marked improvement of clone characteristics. One of the anti-Rh(D)-producing EBV-transformed clones was used to produce an anti-Rh(D) typing reagent which has proved satisfactory for 2 years in routine blood typing in several laboratories. Key words: Monoclonal antibody, human; Blood group antigen; Epstein-Barr virus
Introduction
The study described in this paper was initiated several years ago when our group and others, using murine immunization and hybridization, failed to obtain murine monoclonal antibodies to the Rh(D) antigen with the same specificity as human allo antibodies. The immortalization by Epstein-Barr virus of B lymphocytes producing antibody to several antigens including Rh(D) (Steinitz et al., 1977, 1980; Zurawski et al., 1978; Kozbor et al., 1979; BoylCorrespondence to: D. Goossens, Centre National de R~frrence pour les Groupes Sanguins (CNRGS), 53 boulevard Diderot, 75571 Paris Cedex 12, France. Abbreviations: EBV, Epstein-Barr virus; EBNA, EpsteinBarr nuclear antigen; PVP, polyvinyl pyrrolidone.
ston et al., 1980; Koskimies, 1980; Crawford et al., 1983) have indicated the potential of this technique. The following is a description of our experience over 4 years of producing monoclonal antibodies against antigens of Rh, Kell and A blood groups. It illustrates the fact that the EBV transformation technique, in spite of existing drawbacks, can be most useful for generating stable clones secreting monoclonal antibodies at levels adequate for scientific purposes and medical applications. Materials and methods JDol'lOrS
Lymphocytes were obtained from: (a) Buffy-coats from four hyperimmunized
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
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plasmapheresis donors (E.Q., B.H., P.V. and M.T.) with anti-Rh(D) antibodies, one (R.K.) with antiRh(E) and one (P.P.) with anti-A and -B antibodies. (b) Spleens of two patients: one (S.O.), suffering from thalassemia and sickle cell anemia, had antibodies to Rh(c), Kell, Fy(a) and Jk(b) antigens, the other (A.M.) was splenectomized for a portal hypertension syndrome and had anti-Rh(E) and anti-Kell antibody.
Mononuclear cells Mononuclear cells were isolated from buffy coats or spleen cell suspensions by centrifugation on Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden). Depletion of T lymphocytes This was effected by rosetting with sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (Serva, Heidelberg, F.R.G.) followed by separation on Ficoll-Paque. EB V transformation The B95-8 substrain (gift of Dr. J.M. Bechet, Institut Pasteur) was used for the transformation of B lymphocytes. Two approaches were used in succession. Transformation was effected on 'bulk cultures' as follows: lymphocytes were suspended at 1 × 106 cells/ml in RPMI 1640 with 20% heatinactivated fetal calf serum, L-glutamine 2 raM, penicillin 100 U/ml, streptomycin 100 #g/ml, amphotericin B 0.25/~g/ml (reagents from Gibco, Europe), and were then infected with 200 /~1 virus-containing supernatant/ml. Cells were cultured in 24-well plates (Falcon, Becton Dickinson, ref. 3047) at 37°C in a 5% CO 2 atmosphere. A second type of experiment was conducted with lower cell concentrations ranging from 105 to 102 cells/well ('dilution cultures'), analogous to the limiting dilution technique described by Winger et al. (1983), the cultures being prepared in the same culture medium as above, in 96-well Microtest II or III plates (Falcon, refs. 3042 and 3072), with 105 feeder cells/well (peripheral blood mononuclear cells irradiated at 4000 rad). In these experiments EBV infection was achieved by a prior 2 h incubation of cells in B95-8 supernatant, followed by washing, cell dilution, and plating. In
parallel experiments, Greiner plates (24 wells with 16 subdivisions ref. G-2416, Greiner et fils, Bischwiller, France) were also used, with 10 6 cells/well. Cell transformation was confirmed by the presence of EBNA detected by indirect complement fixation immunofluorescence. (Reedman and Klein, 1973).
Specific antibody detection Supernatants were assayed for specific antiblood group antibodies after the cultures were established. Screening was performed using an agglutination technique, on red blood cells of appropriate phenotype (by papain microtube or antiglobulin test). Specificity was established by testing antibodies against a panel of red blood cells of different phenotypes using the same techniques. Specific antibody concentration Anti-Rh(D) concentrations were estimated in an auto-analyzer using the bromelain PVP and polybrene citrate techniques (Moore, 1980). For anti-D and other antibodies, titers were measured in doubling dilutions on native or papain-treated red blood cells or by the antiglobulin test. Immunoglobulin concentration Immunoglobulin concentrations in supernatants was measured by ELISA, using rabbit anti-human IgG-gamma chain (Dakopatts) and rabbit anti-human IgG (gamma chain-specific) alkaline phosphatase conjugate (Behring). Cloning The cell lines with the highest antibody production were cloned by limiting dilution at concentrations of 40 to 1 cell(s)/well, on Microtest II or III plates, on a feeder layer of human peripheral blood mononuclear cells irradiated at 4000 rad (105 cells/well). Supernatants from wells with growing clones were tested by the above-mentioned techniques. Clones synthesizing specific antibody were transferred to 24-well Falcon plates after 4 weeks of growth and subsequently subcloned. Determination of immunoglobulin class and subclass in cytoplasm and supernatant Cytoplasmic immunoglobulins were char-
195
acterized by direct immunofluorescence with anti-class (IgM, IgG, IgA) and subclass (~'1, 72, 73, 74)-specific FITC-conjugated antiglobulins (Institut Pasteur, France; Cappel, U.S.A.; Dakopatts, Denmark, and the Netherlands Red Cross). The class and subclass of antibodies in the supernatants were determined by (a) indirect immunofluorescence on red blood cells sensitized by the antibody to be tested, with anti-class-specific antiglobulins and (b) an antiglobulin agglutination test using specific anti-subclass immunoglobulins (Netherlands Red Cross). Cell fines used as fusion partners The fusion partners used were: (1) Human lymphoblastoid B-cell line KR-4 (6-thioguanine resistant, ouabain 5 × 10 -4 M resistant), obtained by Kozbor et al. (1982a) and kindly supplied by Dr. John C. Roder (Queen's University, Kingston, Ontario, Canada). (2) Mouse myeloma cell line X63-Ag8.653 (Kearney et al., 1979).
Fusion Fusion was achieved using the technique of Kozbor et al. (1982a). Analysis of cellular nuclear DNA content This was kindly undertaken by M. Kornprobst (INSERM U 181) using flow cytometry. Karyotypes Karyotypes were generously determined by Dr. C. Turleau and Prof. J. de Grouchy (CNRS, Paris, France) and by Prof. Bou6 (Centre International de l'Enfance, INSERM, Paris, France).
Results
Establishment of lymphoblastoid cell lines secreting antibody against Rh(D), Rh(G), Rh(c), Rh(E), Kell and A blood groups Transformation of B lymphocytes was achieved in 'bulk' culture conditions, in Greiner plates or in
TABLE I DETAILS OF VARIOUS ANTIBODY-PRODUCING CLONES STUDIED Transformation conditions
Number of lines cloned
Lines obtained yielding specific antibody-producing clones
Bulk culture
33
QA3
Greiner cultures
21
Dilution cultures 5 x 104
67
104
103
f Exp. 1 Exp. 2
Percentage of specific clones at limiting-dilution cloning a
H2D5
0 53 10
H2218 P1A2 P1A4
25 22 23
H1Gll H2G11 V2Bll V3D9 MBG9 MCG8 RaEll
10 100 100 10 20 100 25
K2Ell T1C10 MIDll
50 40 100 5
T6D2 T8B2
100 100
[ Exp. 1 Exp. 2
a These clones were obtained at cell concentrations ranging from 1 to 10 cells/well.
196
microculture plates at lower cell densities. The transformed cell lines produced antibody with different frequencies according to the donor, and the antibody specificity of a given donor (for example, the anti-Kell frequency for donor AM was much lower than the anti-Rh(E) frequency). The results with peripheral blood lymphocytes were, on occasion as high as those with spleen cells. Cloning: the cell lines with the highest specific antibody production were selected for cloning by limiting dilution. Cloning efficiency was variable depending on the lines and was usually low. There was a greater frequency of lines yielding specific antibody-producing clones among those derived by limiting dilution transformation, though there was also a high frequency (21%) of lines yielding no clonal growth. Furthermore, six of these cell lines proved to be already cloned by the transformation stage (Table I).
Characterization of antibody to Rh, Kell and A antigens Class and subclass. The clones were tested for class and subclass specificities and as shown in
Table II the anti-Rh(D), Rh(G), Rh(c) and Rh(E) antibodies were mostly of IgG1 class. However, there was one IgG 2 anti-Rh(D), two IgM antiRh(D) and one IgM anti-Rh(E) clone. The antiKell antibody was also IgG~. The anti-A and A 1 antibodies were of IgA and IgM class respectively. Antibody specificity was studied by testing a panel which included erythrocytes with deleted and rare phenotypes. Results for four antiRh(D)-producing clones are given in Table III. Antibody specificities were determined in a similar manner for clones reacting with the other blood group antigens, Rh(G), Rh(c), Rh(E), Kell, A and A 1 (data not shown). The anti-Rh and Kell antibodies were active in the indirect antiglobulin tests, and the anti-Rh antibody was active with enzyme treated red cells. The IgM antibodies were active in the saline test. Anti-A and A 1 antibodies P1A2 and P1A4 were active in both the saline and enzyme tests. Antibody production levels. Antibody production varied according to the clone. Mean antibody levels in the supernatants and the hemagghitination titers are shown in Fig. 1. The mean values
TABLE II SPECIFICITY AND CLASS OF THE ANTIBODIES PRODUCED BY THE CLONED EBV TRANSFORMED LINES Lines from which antibody-producing clones were obtained
Donor
Antibody specificity
Class, subclass and light chain
B lymphocyte origin
QA3 H2D5 H2218 H1Gll
E.Q. B.H. B.H. B.H.
Anti-Rh(D) Anti-Rh(D) Anti-Rh(D) Anti-Rh(D)
IgG1, ~ IgG1, ~ IgG1, x IgM, K
Peripheral blood Peripheral blood Peripheral blood Peripheral blood
V2B11 T1C10 T6D2 T8B2
P.V. M.T. M.T. M.T.
Anti-Rh(D) Anti-Rh(D) Anti-Rh(D) Anti-Rh(D)
IgG1, x IgM, ~ IgG2, 2~ IgG1, K
Peripheral Peripheral Peripheral Peripheral
H2Gll
B.H.
Anti-Rh(G)
IgG1, ?,
Peripheral blood
RaEll
S.O.
Anti-Rh(c)
IgGl, ?~
Spleen
K2Ell MBG9 MCG8
R.K. A.M. A.M.
Anti-Rh(E) Anti-Rh(E) Anti-R_h(E)
IgM, K IgG1, K IgG1, ?~
Peripheral blood Spleen Spleen
MIDll
A.M.
Anti-Kell
IgG1, 2~
Spleen
P1A2 P1A4
P.P. P.P.
Anti-A Anti-A 1
IgA, x lgM, ~,
Peripheral blood Peripheral blood
blood blood blood blood
197 TABLE Ill TITER A N D SCORE ~BETWEEN PARENTHESES) ON PAPAIN-TREATED RED CELLS OF ANTIBODY DERIVED F R O M F O U R CLONES Red cell phenotype
Clone QA37C3
---/--D - -/D - Dc - / D c D]v(c) - / D w ( C )
-
~N/~N D'~+ce/dce D v Ce/dce D vl C e / d c e ddccee ddCcee Dccee DccEE DCCee DCWcee
H2D5D2
H2218C5
H1GllD6
o (o)
o (o)
o (o)
o (o)
128 (63) 128 (65) 32 (53)
128 (68) 64 (55) 256 (70)
256 (68) 32 (48) 32 (37)
1 024 (78) 256 (78) 1 024 (85)
64 (61) NT a 64 (50) 0 (0) 0 (0) 0 (0) 64 (48) 128 (58) 64 (53) 64 (55)
128 (66) 32 (36) 256 (71) 0 (0) 0 (0) 0 (0) 128 (65) 128 (73) 128 (71) 128 (53)
32 (43) 16 (36) 123 (57) 0 (0) 0 (0) 0 (0) 64 (58) 64 (60) 64 (60) 64 (61)
256 (73) 64 (55) 256 (78) 128(66) 4(15) b 2 (7) 1 024 (88) 512 (80) 256 (80) 256 (75)
a Not tested. b Antibody from H 1 D l l did not react with Rh(D) negative red cells in saline but gave a slight reaction with the same cells when papain treated.
ranged from 4 # g / m l (clones from QA3) to 50 /xg/ml (clones from MIDII) with a mean value of 25/t g/rnl. Stability of growth and antibody production. The clones described in Fig. 1 have been growing in continuous culture from 1 to 4 years. With the Cat[ Line ar~-I~(O)
QA3
Duration of contin¢~.~ Culture ~ ~years 3 years 2 years ( 3 months)" 2 years
J I I I
MC68
2 years 1,S year 1 year 1 year
} knd [ I
alfi-Ket[ MID11 anti-A P1A2 anti- A1 P1A~,
1 year {1 year)2 years
I
H2OS H2218
H1r;'11 ~nh-Rh(O) H2611 • ~ti-Rhff) ReEl1 ~ f i - Rh(E) K2Ell MBF'9
Antibody Concentration ( ~ g / m l ) 2p t~ 6p
r---, =
~ ~
exception of clones from H 1 G l l and P1A2, one or more clones derived from each line have been stable in growth and antibody production. However, not all clones derived from a given line evolved in an identical fashion. For example, one of the five QA3 clones in culture lost its capacity to produce light chain after 26 months of growth (as shown by immunofluorescence tests) while the others still produce anti-Rh(D). A karyotype analysis in the laboratory of Professor Bou~ did not show any chromosomal loss.
'
,
"'t i
i-----I ~ +
+
[DO }nd i
.
.
.
.
i
.
5
0
.
.
.
i
,
•
,
t
,
10
Log 2 HemaggtutinationTiter
Fig. 1. Antibody production by 12 antibody levels. For each cell line concentration and the lower bar * Clones H 1 G l l and P1A2 could longer periods of time.
clones: duration and mean the upper bar is antibody is hemagglutination titer. not be kept in culture for
!
5
|0
15
moBths
Fig. 2. Evolution of anti-D production by clone H2D5D2F5 over a period of 14 months (from June 1984 to July 1985).
198
Clones from H1G11 and P1A2 could not maintain growth in culture and after 3 months and 1 year respectively, cell division slowed and then stopped. Fig. 2 illustrates the evolution of antibody production over a period of 14 months for clone H2D5D2F5. Antibody levels in the supernatant changed but the mean levels remained relatively stable.
Fusion of anti-Rh(D) producing lymphoblastoid clones with human or mouse hybridization partners Hybridization with two different partners was attempted since it was possible that this procedure would increase antibody production, an effect which would have been useful with clones such as those derived from line QA3. It was also hoped that this would increase the cloning efficiency (Kozbor et al., 1982a, b). Fusion with KR-4. Fusions were achieved between anti-D-producing clones QA37C3 or H2D5D2 and the human lymphoblastoid line KR4. In both cases all of the hybrids obtained secreted anti-D antibody of the same class and subclass as the parent EBV clones. The level of specific antibody production by the hybrids was not significantly different from the parent clone antibody production, the mean antibody levels for eight hybrids over 3 months ranging from 1.2 _+0.2 to 4.9 _+_1.5 #g/ml, with a mean level for QA37C3 of 4 _+ 2.1 /~g/rnl. The cloning efficiency of the hybrids was similar to that of parent lymphoblastoid lines. Two clones were kept in continuous culture for I year, and exhibited stable antibody production. The hybrid nature of these cells was supported by the fact that the parent cells did not survive in selective medium, and by the results of measurement of the DNA content by flow cytometry. The latter revealed that cells of the hybrid clones had peak fluorescence intensities which indicated a doubled DNA content compared to the parent lines. Karyotype analysis also showed that the hybrid cells were tetraploid or near tetraploid (from 86 to 92 chromosomes). Furthermore four 'marker' chromosomes present in KR-4 cells were also observable in these cells, demonstrating their derivation from KR-4. Fusion with mouse myeloma X63-Ag8.653. This myeloma proved an efficient fusion partner in an
TABLE IV RESULTS OF A MULTI-CENTER STUDY OF 19 LABORATORIES ON Rh(D) TYPING WITH A MONOCLONAL REAGENT DERIVED FROM CLONE H2D5D2
Rh(D) positive Weak Rh(D) b Rh(D) negative
Number of individuals tested a
Number positive with standard reagent
Number positive with H2D5D2
7 926 199 1094
7 926 0 0
7 926 37 0
Typingby a slide test and compared to that observed with an anti-Rh(D) reagent used in each laboratory. b Weak Rh(D) was defined as Rh(D) antigen detectable by Coombs technique or using an auto-analyzer,and not by a slide test with a standard reagent. a
experiment undertaken with a subclone of H2D5D2. One of the hybrids yielded a clone (1C5E2) with an antibody concentration of 20 #g/ml, almost twice that of the parent (12/~g/ml) but others showed antibody production equivalent to the parent clone. Clone 1C5E2 has secreted anti-Rh (D) for 16 months while 15 others cultured simultaneously lost specific antibody production over a 1-6 month period.
Production of high quantities of anti-Rh(D) and Rh(c) containing supernatants Experiments were performed in order to verify whether the clones from lymphoblastoid cell lines could adapt in high volume culture conditions while maintaining their antibody production at a satisfactory level. Cells from an anti-D-producing subclone of H2D5 were progressively grown up from 10 ml cultures to 500 ml cultures in roller bottles. Cells were grown in Iscove's modified Dulbecco's medium (IMDM) with 3% FCS, in closed bottles in a Bellco roller incubator. The maximum cell concentration attained ranged from 3-5 x 106 cells/ml compared to 1.5-2.5 x 106 in flasks, and antibody concentrations reached in the roller bottle system (after 7 days of culture with the cells being 'seeded' at 0.5 x 106 cells/ml) were 25 + 5 /~g/ml as measured by an auto-analyzer. The antiRh(c) producing clone RaE112-B8 was grown un-
199
der the same conditions and yielded supernatants with a mean antibody concentration of 20 ~tg/ml.
Use of supernatant to produce anti-Rh(D) and antiRh(c) typing reagents Supernatants from the H2D5 subclone were used to make a reagent containing 15 ~tg/ml of anti-D in albumin at a concentration of 150 g/1. This reagent could be used in the slide test and tube test techniques and was also suitable for automated machines such as the Auto-analyzer and the Groupamatic. A study of a monoclonal reagent in routine blood typing, involving 19 transfusion centers in France was performed in parallel with standard polyclonal reagents using both manual techniques (Table IV) and the Groupamatic analyzer (data not shown) on 9219 and 7943 blood samples respectively. Identical results were observed in reactions with the common Rh phenotypes and 18% more Rh(D)-positive subjects with weak Rh antigens were detected by the monoclonal reagent in the manual techniques. Supernatant from the anti-Rh(c)-producing clone RaEll2-B8 is now also being used to produce a typing reagent and anti-Rh(E) and anti-Kell antibodies are now being tested as potential reagents.
Discussion
In this study, we have used the technique of EBV transformation to establish human monoclonal B cell lines secreting antibody directed against blood group antigens. We have obtained a number of clones which are stable (some for at least 4 years) and whose antibody production is in some cases high enough to permit their use for the production of typing reagents. The EBV transformations were induced in B lymphocyte populations obtained from hyperimmunized plasmapheresis donors or the spleen cells of immunized patients. The interval between the last restimulation of the donor and the transformation experiments ranged from 1 to 18 months, which was much longer than that used by Kozbor and Roder (1981) but still allowed the establishment of numerous
specific cell lines. This was probably because of the hyperimmunized state of the donors. The frequency of specific antibody-producing lines varied from one donor to another and according to the specificities for a given donor. EBV transformation was effected either in 'bulk' cultures or at lower cell concentrations. Seeding EBV-infected cells at low concentrations (10 3 tO 5 × 10 4) increased the frequency of 'precloned' cell lines and facilitated isolation of specific antibody producing clones. Clones producing antibody of the three major immunoglobulin classes were produced with a distribution consistent with the observed frequency of each class for a given specificity. The antibody specificity of these clones was clearly defined in most cases, following the reaction pattern of human sera. In our experience these clones have been stable, at least one clone of each group having been grown continuously for periods of 1-4 years without further subcloning. The level of antibody production was different from one clone to another and was subject to oscillations for a given clone. However, the mean level of production remained stable with no tendency to decrease, over long periods of time. Several EBV-transformed clones underwent a decline either in antibody production or in growth. In the first case, the defect was due to a loss of light or heavy chain production in a part of the cell population. This deterioration does not usually affect more than one clone of a given line, and can be avoided by regular monitoring of the clones for antibody production and for class and type of cytoplasmic immunoglobulin, followed by subcloning when necessary. A more difficult problem is that of B cell clones with a low capacity for growth such as H 1 G l l or PIA2, which can only be kept in culture for short periods of time, a characteristic similar to that described by Melamed et al. (1985). Rescue of these lines may be possible by hybridization of cells frozen at an early stage of the culture. Hybridization of two anti-Rh(D)-producing clones with the human lymphoblastoid line KR-4 resuited in stable hybrids but did not improve antibody production or cloning efficiency. Fusion with mouse myeloma X63-Ag8.653 resulted in essentially unstable hybrids, except for one hybrid which was still stable 16 months later and secreted anti-
200
Rh(D) at a level almost double that of the parent clone. We have been interested in developing selected clones to produce large quantities of anti-Rh antibodies by culture in a roller bottle system. Our aim has been to provide a reagent for blood typing and to supply a source of purified monoclonal antibody for biochemical studies of the Rh antigens. At present, these antibodies are being used to purify Rh(D) (Bloy et al., 1987), -(c) and -(G) antigens from the red cell membrane and to study their inter-relationships. This project is under way in the laboratories of J.P. Cartron and C. Doinel at the C.N.T.S. Institut. A typing reagent produced from a subclone of H2D5 has been shown to be specific and efficient in Rh(D) blood group typing, first in a preliminary study undertaken by 19 transfusion centers and since then, for a period of more than 1 year, by the routine use of the reagent by many laboratories. The establishment of appropriate production and purification sequences and quality controls are now in progress with a view to developing a therapeutic preparation for the prevention of hemolytic disease of the newborn due to Rh(D) allo immunization.
Acknowledgements We thank Dr. B. Mercier, Dr. M.C. DupuyMontbrun and Dr. D. Lyon-Caen for their cooperation in providing buffy coats from immunized donors, P. Gane for the immunofluorescence tests, and V. Fayet for the preparation of this manuscript. This work was supported by grants from INSERM (National Institute for Health and Medical Research), the Pierre et Marie Curie University, Paris, France, and from CNAM (Caisse Nationale d'Assurance Maladie). References Bloy, C., Blanchard, D., Lambin, P., Goossens, D., Rouger, Ph., Salmon, Ch. and Cartron, J.P. (1987) Human monoclonal antibodies against Rh antigens: partial characterization of the Rh(D) polypeptide from human erythrocytes, submitted. Boylston, A.W., Gardner, B., Anderson, R.L. and HughesJones, N.C. (1980) Production of the human IgM anti-D in
tissue culture by EB-Vims transformed lymphocytes. Scand. J. Immunol. 12, 355. Crawford, D.H., Harrison, J.F., Barlow, M.J., Winger, L. and Huehns, E.R. (1983) Production of human monoclonal antibody to Rhesus D antigen. Lancet 19, 386. Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewski, K., (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody secreting hybrid cell lines. J. Immunol. 123, 1548. Koskimies, S. (1980) Human lymphoblastoid cell line producing antibody against Rh antigen D. Scand. J. Immunol. 11, 73. Kozbor, D. and Roder, J.C. (1981) Requirements for the establishment of high-titered human monoclonal antibodies against tetanus toxoid using the Epstein-Barr virus technique. J. Immunol. 127, 1275. Kozbor, D., Steinitz, M., Klein, G., Koskimies, S. and Makela, O, (1979) Establishment of anti-TNP antibody producing human lymphoid lines by preselection for hapten binding followed by EBV transformation. Scand. J. Immunol. 10, 187. Kozbor, D., Lagarde, H.E. and Roder, J.C. (1982a) Human hybridoma constructed with antigen specific Epstein-Barr virus transformed lines. Proc. Natl. Acad. Sci. U.S.A. 79, 6651. Kozbor, D., Roder, J.C., Chang, M., Steplewski, Z. and Koprowski, H. (1982b) Human anti-tetanus toxoid monoclonal antibody secreted by EBV-transformed human B cells fused with murine myeloma. Hybridoma 1,323. Melamed, M.D., Gordon, J., Ley, S.J., Edgar, D. and HughesJones, N.C. (1985) Senescence of a human lymphoblastoid clone producing anti-Rhesus (D). Eur. J. Immunol. 15,742. Moore, B.P.L (Ed.) (1980) Serological and immunological methods of the Canadian Red Cross Blood Transfusion Service, 8th edn. (Toronto, Ontario). Olsson, L. and Kaplan, H.S. (1980) Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity. Proc. Natl. Acad. Sci. U.S.A., 77, 5429. Reedman, B.M. and Klein, G. (1973) Cellular localization of an Epstein-Barr virus (EBV) associated complement fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11,499. Steinitz, M., Klein, G., Koskimies, S. and Makela, O, (1977) EB-virus induced B lymphocyte cell lines producing specific antibody. Nature 269, 420. Steinitz, M., Izak, G., Cohen, S., Ehrenfeld, M. and Flechner, I. (1980) Continuous production of monoclonal rheumatoid factor by EBV transformed lymphocytes. Nature 287, 443. Vindelov, L. (1977) Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. Virchows Arch. B. Cell. Pathol. 24, 227. Winger, L., Winger, C., Shastry, P., Russell, A. and Longenecker, M. (1983) Efficient generation in vitro from human peripheral blood cells of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization. Proc. Natl. Acad. Sci. U.S.A. 80, 4484. Zurawski, Jr., V.R., Haber, E. and Black, P.H. (1978) Production of antibody to tetanus toxoid by continuous human lymphoblastoid cell lines. Science 199, 1439.
193
Elsevier JIM 04408
Human monoclonal antibodies against blood group antigens Preparation of a series of stable EBV immortalized B clones producing high levels of antibody of different isotypes and specificities Dominique Goossens 1, Fran~oise Champomier 1, Philippe Rouger 2 and Charles Salmon 2 i Centre National de Rkfkreneepour les Groupes Sanguins, CNTS Institut, Paris, and e Universit~ Pierre et Marie Curie, 75571 Paris Cedex 12, France
(Received 24 November 1986, revised received 22 January 1987, accepted 17 March 1987)
The EBV immortalization technique was used to produce stable clones, from B lymphocytes, secreting human monoclonal antibodies to Rh(D), Rh(G), Rh(c), Rh(E), Kell, A and A 1 blood group antigens. These clones were obtained from peripheral blood lymphocytes of hyperimmunized plasmapheresis donors or from spleen lymphocytes of immunized patients. Mean levels of antibody concentration varied between 4 and 50 #g/ml. The antibodies obtained were of IgG~, IgG2, IgM or IgA class. Most of the clones have been stable for growth and antibody production during long periods of continuous culture, extending upto 4 years. Hybridization of two clones was effected with the human lymphoblastoid cell line KR-4 and with the mouse myeloma X63-Ag8.653, but did not result in any marked improvement of clone characteristics. One of the anti-Rh(D)-producing EBV-transformed clones was used to produce an anti-Rh(D) typing reagent which has proved satisfactory for 2 years in routine blood typing in several laboratories. Key words: Monoclonal antibody, human; Blood group antigen; Epstein-Barr virus
Introduction
The study described in this paper was initiated several years ago when our group and others, using murine immunization and hybridization, failed to obtain murine monoclonal antibodies to the Rh(D) antigen with the same specificity as human allo antibodies. The immortalization by Epstein-Barr virus of B lymphocytes producing antibody to several antigens including Rh(D) (Steinitz et al., 1977, 1980; Zurawski et al., 1978; Kozbor et al., 1979; BoylCorrespondence to: D. Goossens, Centre National de R~frrence pour les Groupes Sanguins (CNRGS), 53 boulevard Diderot, 75571 Paris Cedex 12, France. Abbreviations: EBV, Epstein-Barr virus; EBNA, EpsteinBarr nuclear antigen; PVP, polyvinyl pyrrolidone.
ston et al., 1980; Koskimies, 1980; Crawford et al., 1983) have indicated the potential of this technique. The following is a description of our experience over 4 years of producing monoclonal antibodies against antigens of Rh, Kell and A blood groups. It illustrates the fact that the EBV transformation technique, in spite of existing drawbacks, can be most useful for generating stable clones secreting monoclonal antibodies at levels adequate for scientific purposes and medical applications. Materials and methods JDol'lOrS
Lymphocytes were obtained from: (a) Buffy-coats from four hyperimmunized
0022-1759/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
194
plasmapheresis donors (E.Q., B.H., P.V. and M.T.) with anti-Rh(D) antibodies, one (R.K.) with antiRh(E) and one (P.P.) with anti-A and -B antibodies. (b) Spleens of two patients: one (S.O.), suffering from thalassemia and sickle cell anemia, had antibodies to Rh(c), Kell, Fy(a) and Jk(b) antigens, the other (A.M.) was splenectomized for a portal hypertension syndrome and had anti-Rh(E) and anti-Kell antibody.
Mononuclear cells Mononuclear cells were isolated from buffy coats or spleen cell suspensions by centrifugation on Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden). Depletion of T lymphocytes This was effected by rosetting with sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (Serva, Heidelberg, F.R.G.) followed by separation on Ficoll-Paque. EB V transformation The B95-8 substrain (gift of Dr. J.M. Bechet, Institut Pasteur) was used for the transformation of B lymphocytes. Two approaches were used in succession. Transformation was effected on 'bulk cultures' as follows: lymphocytes were suspended at 1 × 106 cells/ml in RPMI 1640 with 20% heatinactivated fetal calf serum, L-glutamine 2 raM, penicillin 100 U/ml, streptomycin 100 #g/ml, amphotericin B 0.25/~g/ml (reagents from Gibco, Europe), and were then infected with 200 /~1 virus-containing supernatant/ml. Cells were cultured in 24-well plates (Falcon, Becton Dickinson, ref. 3047) at 37°C in a 5% CO 2 atmosphere. A second type of experiment was conducted with lower cell concentrations ranging from 105 to 102 cells/well ('dilution cultures'), analogous to the limiting dilution technique described by Winger et al. (1983), the cultures being prepared in the same culture medium as above, in 96-well Microtest II or III plates (Falcon, refs. 3042 and 3072), with 105 feeder cells/well (peripheral blood mononuclear cells irradiated at 4000 rad). In these experiments EBV infection was achieved by a prior 2 h incubation of cells in B95-8 supernatant, followed by washing, cell dilution, and plating. In
parallel experiments, Greiner plates (24 wells with 16 subdivisions ref. G-2416, Greiner et fils, Bischwiller, France) were also used, with 10 6 cells/well. Cell transformation was confirmed by the presence of EBNA detected by indirect complement fixation immunofluorescence. (Reedman and Klein, 1973).
Specific antibody detection Supernatants were assayed for specific antiblood group antibodies after the cultures were established. Screening was performed using an agglutination technique, on red blood cells of appropriate phenotype (by papain microtube or antiglobulin test). Specificity was established by testing antibodies against a panel of red blood cells of different phenotypes using the same techniques. Specific antibody concentration Anti-Rh(D) concentrations were estimated in an auto-analyzer using the bromelain PVP and polybrene citrate techniques (Moore, 1980). For anti-D and other antibodies, titers were measured in doubling dilutions on native or papain-treated red blood cells or by the antiglobulin test. Immunoglobulin concentration Immunoglobulin concentrations in supernatants was measured by ELISA, using rabbit anti-human IgG-gamma chain (Dakopatts) and rabbit anti-human IgG (gamma chain-specific) alkaline phosphatase conjugate (Behring). Cloning The cell lines with the highest antibody production were cloned by limiting dilution at concentrations of 40 to 1 cell(s)/well, on Microtest II or III plates, on a feeder layer of human peripheral blood mononuclear cells irradiated at 4000 rad (105 cells/well). Supernatants from wells with growing clones were tested by the above-mentioned techniques. Clones synthesizing specific antibody were transferred to 24-well Falcon plates after 4 weeks of growth and subsequently subcloned. Determination of immunoglobulin class and subclass in cytoplasm and supernatant Cytoplasmic immunoglobulins were char-
195
acterized by direct immunofluorescence with anti-class (IgM, IgG, IgA) and subclass (~'1, 72, 73, 74)-specific FITC-conjugated antiglobulins (Institut Pasteur, France; Cappel, U.S.A.; Dakopatts, Denmark, and the Netherlands Red Cross). The class and subclass of antibodies in the supernatants were determined by (a) indirect immunofluorescence on red blood cells sensitized by the antibody to be tested, with anti-class-specific antiglobulins and (b) an antiglobulin agglutination test using specific anti-subclass immunoglobulins (Netherlands Red Cross). Cell fines used as fusion partners The fusion partners used were: (1) Human lymphoblastoid B-cell line KR-4 (6-thioguanine resistant, ouabain 5 × 10 -4 M resistant), obtained by Kozbor et al. (1982a) and kindly supplied by Dr. John C. Roder (Queen's University, Kingston, Ontario, Canada). (2) Mouse myeloma cell line X63-Ag8.653 (Kearney et al., 1979).
Fusion Fusion was achieved using the technique of Kozbor et al. (1982a). Analysis of cellular nuclear DNA content This was kindly undertaken by M. Kornprobst (INSERM U 181) using flow cytometry. Karyotypes Karyotypes were generously determined by Dr. C. Turleau and Prof. J. de Grouchy (CNRS, Paris, France) and by Prof. Bou6 (Centre International de l'Enfance, INSERM, Paris, France).
Results
Establishment of lymphoblastoid cell lines secreting antibody against Rh(D), Rh(G), Rh(c), Rh(E), Kell and A blood groups Transformation of B lymphocytes was achieved in 'bulk' culture conditions, in Greiner plates or in
TABLE I DETAILS OF VARIOUS ANTIBODY-PRODUCING CLONES STUDIED Transformation conditions
Number of lines cloned
Lines obtained yielding specific antibody-producing clones
Bulk culture
33
QA3
Greiner cultures
21
Dilution cultures 5 x 104
67
104
103
f Exp. 1 Exp. 2
Percentage of specific clones at limiting-dilution cloning a
H2D5
0 53 10
H2218 P1A2 P1A4
25 22 23
H1Gll H2G11 V2Bll V3D9 MBG9 MCG8 RaEll
10 100 100 10 20 100 25
K2Ell T1C10 MIDll
50 40 100 5
T6D2 T8B2
100 100
[ Exp. 1 Exp. 2
a These clones were obtained at cell concentrations ranging from 1 to 10 cells/well.
196
microculture plates at lower cell densities. The transformed cell lines produced antibody with different frequencies according to the donor, and the antibody specificity of a given donor (for example, the anti-Kell frequency for donor AM was much lower than the anti-Rh(E) frequency). The results with peripheral blood lymphocytes were, on occasion as high as those with spleen cells. Cloning: the cell lines with the highest specific antibody production were selected for cloning by limiting dilution. Cloning efficiency was variable depending on the lines and was usually low. There was a greater frequency of lines yielding specific antibody-producing clones among those derived by limiting dilution transformation, though there was also a high frequency (21%) of lines yielding no clonal growth. Furthermore, six of these cell lines proved to be already cloned by the transformation stage (Table I).
Characterization of antibody to Rh, Kell and A antigens Class and subclass. The clones were tested for class and subclass specificities and as shown in
Table II the anti-Rh(D), Rh(G), Rh(c) and Rh(E) antibodies were mostly of IgG1 class. However, there was one IgG 2 anti-Rh(D), two IgM antiRh(D) and one IgM anti-Rh(E) clone. The antiKell antibody was also IgG~. The anti-A and A 1 antibodies were of IgA and IgM class respectively. Antibody specificity was studied by testing a panel which included erythrocytes with deleted and rare phenotypes. Results for four antiRh(D)-producing clones are given in Table III. Antibody specificities were determined in a similar manner for clones reacting with the other blood group antigens, Rh(G), Rh(c), Rh(E), Kell, A and A 1 (data not shown). The anti-Rh and Kell antibodies were active in the indirect antiglobulin tests, and the anti-Rh antibody was active with enzyme treated red cells. The IgM antibodies were active in the saline test. Anti-A and A 1 antibodies P1A2 and P1A4 were active in both the saline and enzyme tests. Antibody production levels. Antibody production varied according to the clone. Mean antibody levels in the supernatants and the hemagghitination titers are shown in Fig. 1. The mean values
TABLE II SPECIFICITY AND CLASS OF THE ANTIBODIES PRODUCED BY THE CLONED EBV TRANSFORMED LINES Lines from which antibody-producing clones were obtained
Donor
Antibody specificity
Class, subclass and light chain
B lymphocyte origin
QA3 H2D5 H2218 H1Gll
E.Q. B.H. B.H. B.H.
Anti-Rh(D) Anti-Rh(D) Anti-Rh(D) Anti-Rh(D)
IgG1, ~ IgG1, ~ IgG1, x IgM, K
Peripheral blood Peripheral blood Peripheral blood Peripheral blood
V2B11 T1C10 T6D2 T8B2
P.V. M.T. M.T. M.T.
Anti-Rh(D) Anti-Rh(D) Anti-Rh(D) Anti-Rh(D)
IgG1, x IgM, ~ IgG2, 2~ IgG1, K
Peripheral Peripheral Peripheral Peripheral
H2Gll
B.H.
Anti-Rh(G)
IgG1, ?,
Peripheral blood
RaEll
S.O.
Anti-Rh(c)
IgGl, ?~
Spleen
K2Ell MBG9 MCG8
R.K. A.M. A.M.
Anti-Rh(E) Anti-Rh(E) Anti-R_h(E)
IgM, K IgG1, K IgG1, ?~
Peripheral blood Spleen Spleen
MIDll
A.M.
Anti-Kell
IgG1, 2~
Spleen
P1A2 P1A4
P.P. P.P.
Anti-A Anti-A 1
IgA, x lgM, ~,
Peripheral blood Peripheral blood
blood blood blood blood
197 TABLE Ill TITER A N D SCORE ~BETWEEN PARENTHESES) ON PAPAIN-TREATED RED CELLS OF ANTIBODY DERIVED F R O M F O U R CLONES Red cell phenotype
Clone QA37C3
---/--D - -/D - Dc - / D c D]v(c) - / D w ( C )
-
~N/~N D'~+ce/dce D v Ce/dce D vl C e / d c e ddccee ddCcee Dccee DccEE DCCee DCWcee
H2D5D2
H2218C5
H1GllD6
o (o)
o (o)
o (o)
o (o)
128 (63) 128 (65) 32 (53)
128 (68) 64 (55) 256 (70)
256 (68) 32 (48) 32 (37)
1 024 (78) 256 (78) 1 024 (85)
64 (61) NT a 64 (50) 0 (0) 0 (0) 0 (0) 64 (48) 128 (58) 64 (53) 64 (55)
128 (66) 32 (36) 256 (71) 0 (0) 0 (0) 0 (0) 128 (65) 128 (73) 128 (71) 128 (53)
32 (43) 16 (36) 123 (57) 0 (0) 0 (0) 0 (0) 64 (58) 64 (60) 64 (60) 64 (61)
256 (73) 64 (55) 256 (78) 128(66) 4(15) b 2 (7) 1 024 (88) 512 (80) 256 (80) 256 (75)
a Not tested. b Antibody from H 1 D l l did not react with Rh(D) negative red cells in saline but gave a slight reaction with the same cells when papain treated.
ranged from 4 # g / m l (clones from QA3) to 50 /xg/ml (clones from MIDII) with a mean value of 25/t g/rnl. Stability of growth and antibody production. The clones described in Fig. 1 have been growing in continuous culture from 1 to 4 years. With the Cat[ Line ar~-I~(O)
QA3
Duration of contin¢~.~ Culture ~ ~years 3 years 2 years ( 3 months)" 2 years
J I I I
MC68
2 years 1,S year 1 year 1 year
} knd [ I
alfi-Ket[ MID11 anti-A P1A2 anti- A1 P1A~,
1 year {1 year)2 years
I
H2OS H2218
H1r;'11 ~nh-Rh(O) H2611 • ~ti-Rhff) ReEl1 ~ f i - Rh(E) K2Ell MBF'9
Antibody Concentration ( ~ g / m l ) 2p t~ 6p
r---, =
~ ~
exception of clones from H 1 G l l and P1A2, one or more clones derived from each line have been stable in growth and antibody production. However, not all clones derived from a given line evolved in an identical fashion. For example, one of the five QA3 clones in culture lost its capacity to produce light chain after 26 months of growth (as shown by immunofluorescence tests) while the others still produce anti-Rh(D). A karyotype analysis in the laboratory of Professor Bou~ did not show any chromosomal loss.
'
,
"'t i
i-----I ~ +
+
[DO }nd i
.
.
.
.
i
.
5
0
.
.
.
i
,
•
,
t
,
10
Log 2 HemaggtutinationTiter
Fig. 1. Antibody production by 12 antibody levels. For each cell line concentration and the lower bar * Clones H 1 G l l and P1A2 could longer periods of time.
clones: duration and mean the upper bar is antibody is hemagglutination titer. not be kept in culture for
!
5
|0
15
moBths
Fig. 2. Evolution of anti-D production by clone H2D5D2F5 over a period of 14 months (from June 1984 to July 1985).
198
Clones from H1G11 and P1A2 could not maintain growth in culture and after 3 months and 1 year respectively, cell division slowed and then stopped. Fig. 2 illustrates the evolution of antibody production over a period of 14 months for clone H2D5D2F5. Antibody levels in the supernatant changed but the mean levels remained relatively stable.
Fusion of anti-Rh(D) producing lymphoblastoid clones with human or mouse hybridization partners Hybridization with two different partners was attempted since it was possible that this procedure would increase antibody production, an effect which would have been useful with clones such as those derived from line QA3. It was also hoped that this would increase the cloning efficiency (Kozbor et al., 1982a, b). Fusion with KR-4. Fusions were achieved between anti-D-producing clones QA37C3 or H2D5D2 and the human lymphoblastoid line KR4. In both cases all of the hybrids obtained secreted anti-D antibody of the same class and subclass as the parent EBV clones. The level of specific antibody production by the hybrids was not significantly different from the parent clone antibody production, the mean antibody levels for eight hybrids over 3 months ranging from 1.2 _+0.2 to 4.9 _+_1.5 #g/ml, with a mean level for QA37C3 of 4 _+ 2.1 /~g/rnl. The cloning efficiency of the hybrids was similar to that of parent lymphoblastoid lines. Two clones were kept in continuous culture for I year, and exhibited stable antibody production. The hybrid nature of these cells was supported by the fact that the parent cells did not survive in selective medium, and by the results of measurement of the DNA content by flow cytometry. The latter revealed that cells of the hybrid clones had peak fluorescence intensities which indicated a doubled DNA content compared to the parent lines. Karyotype analysis also showed that the hybrid cells were tetraploid or near tetraploid (from 86 to 92 chromosomes). Furthermore four 'marker' chromosomes present in KR-4 cells were also observable in these cells, demonstrating their derivation from KR-4. Fusion with mouse myeloma X63-Ag8.653. This myeloma proved an efficient fusion partner in an
TABLE IV RESULTS OF A MULTI-CENTER STUDY OF 19 LABORATORIES ON Rh(D) TYPING WITH A MONOCLONAL REAGENT DERIVED FROM CLONE H2D5D2
Rh(D) positive Weak Rh(D) b Rh(D) negative
Number of individuals tested a
Number positive with standard reagent
Number positive with H2D5D2
7 926 199 1094
7 926 0 0
7 926 37 0
Typingby a slide test and compared to that observed with an anti-Rh(D) reagent used in each laboratory. b Weak Rh(D) was defined as Rh(D) antigen detectable by Coombs technique or using an auto-analyzer,and not by a slide test with a standard reagent. a
experiment undertaken with a subclone of H2D5D2. One of the hybrids yielded a clone (1C5E2) with an antibody concentration of 20 #g/ml, almost twice that of the parent (12/~g/ml) but others showed antibody production equivalent to the parent clone. Clone 1C5E2 has secreted anti-Rh (D) for 16 months while 15 others cultured simultaneously lost specific antibody production over a 1-6 month period.
Production of high quantities of anti-Rh(D) and Rh(c) containing supernatants Experiments were performed in order to verify whether the clones from lymphoblastoid cell lines could adapt in high volume culture conditions while maintaining their antibody production at a satisfactory level. Cells from an anti-D-producing subclone of H2D5 were progressively grown up from 10 ml cultures to 500 ml cultures in roller bottles. Cells were grown in Iscove's modified Dulbecco's medium (IMDM) with 3% FCS, in closed bottles in a Bellco roller incubator. The maximum cell concentration attained ranged from 3-5 x 106 cells/ml compared to 1.5-2.5 x 106 in flasks, and antibody concentrations reached in the roller bottle system (after 7 days of culture with the cells being 'seeded' at 0.5 x 106 cells/ml) were 25 + 5 /~g/ml as measured by an auto-analyzer. The antiRh(c) producing clone RaE112-B8 was grown un-
199
der the same conditions and yielded supernatants with a mean antibody concentration of 20 ~tg/ml.
Use of supernatant to produce anti-Rh(D) and antiRh(c) typing reagents Supernatants from the H2D5 subclone were used to make a reagent containing 15 ~tg/ml of anti-D in albumin at a concentration of 150 g/1. This reagent could be used in the slide test and tube test techniques and was also suitable for automated machines such as the Auto-analyzer and the Groupamatic. A study of a monoclonal reagent in routine blood typing, involving 19 transfusion centers in France was performed in parallel with standard polyclonal reagents using both manual techniques (Table IV) and the Groupamatic analyzer (data not shown) on 9219 and 7943 blood samples respectively. Identical results were observed in reactions with the common Rh phenotypes and 18% more Rh(D)-positive subjects with weak Rh antigens were detected by the monoclonal reagent in the manual techniques. Supernatant from the anti-Rh(c)-producing clone RaEll2-B8 is now also being used to produce a typing reagent and anti-Rh(E) and anti-Kell antibodies are now being tested as potential reagents.
Discussion
In this study, we have used the technique of EBV transformation to establish human monoclonal B cell lines secreting antibody directed against blood group antigens. We have obtained a number of clones which are stable (some for at least 4 years) and whose antibody production is in some cases high enough to permit their use for the production of typing reagents. The EBV transformations were induced in B lymphocyte populations obtained from hyperimmunized plasmapheresis donors or the spleen cells of immunized patients. The interval between the last restimulation of the donor and the transformation experiments ranged from 1 to 18 months, which was much longer than that used by Kozbor and Roder (1981) but still allowed the establishment of numerous
specific cell lines. This was probably because of the hyperimmunized state of the donors. The frequency of specific antibody-producing lines varied from one donor to another and according to the specificities for a given donor. EBV transformation was effected either in 'bulk' cultures or at lower cell concentrations. Seeding EBV-infected cells at low concentrations (10 3 tO 5 × 10 4) increased the frequency of 'precloned' cell lines and facilitated isolation of specific antibody producing clones. Clones producing antibody of the three major immunoglobulin classes were produced with a distribution consistent with the observed frequency of each class for a given specificity. The antibody specificity of these clones was clearly defined in most cases, following the reaction pattern of human sera. In our experience these clones have been stable, at least one clone of each group having been grown continuously for periods of 1-4 years without further subcloning. The level of antibody production was different from one clone to another and was subject to oscillations for a given clone. However, the mean level of production remained stable with no tendency to decrease, over long periods of time. Several EBV-transformed clones underwent a decline either in antibody production or in growth. In the first case, the defect was due to a loss of light or heavy chain production in a part of the cell population. This deterioration does not usually affect more than one clone of a given line, and can be avoided by regular monitoring of the clones for antibody production and for class and type of cytoplasmic immunoglobulin, followed by subcloning when necessary. A more difficult problem is that of B cell clones with a low capacity for growth such as H 1 G l l or PIA2, which can only be kept in culture for short periods of time, a characteristic similar to that described by Melamed et al. (1985). Rescue of these lines may be possible by hybridization of cells frozen at an early stage of the culture. Hybridization of two anti-Rh(D)-producing clones with the human lymphoblastoid line KR-4 resuited in stable hybrids but did not improve antibody production or cloning efficiency. Fusion with mouse myeloma X63-Ag8.653 resulted in essentially unstable hybrids, except for one hybrid which was still stable 16 months later and secreted anti-
200
Rh(D) at a level almost double that of the parent clone. We have been interested in developing selected clones to produce large quantities of anti-Rh antibodies by culture in a roller bottle system. Our aim has been to provide a reagent for blood typing and to supply a source of purified monoclonal antibody for biochemical studies of the Rh antigens. At present, these antibodies are being used to purify Rh(D) (Bloy et al., 1987), -(c) and -(G) antigens from the red cell membrane and to study their inter-relationships. This project is under way in the laboratories of J.P. Cartron and C. Doinel at the C.N.T.S. Institut. A typing reagent produced from a subclone of H2D5 has been shown to be specific and efficient in Rh(D) blood group typing, first in a preliminary study undertaken by 19 transfusion centers and since then, for a period of more than 1 year, by the routine use of the reagent by many laboratories. The establishment of appropriate production and purification sequences and quality controls are now in progress with a view to developing a therapeutic preparation for the prevention of hemolytic disease of the newborn due to Rh(D) allo immunization.
Acknowledgements We thank Dr. B. Mercier, Dr. M.C. DupuyMontbrun and Dr. D. Lyon-Caen for their cooperation in providing buffy coats from immunized donors, P. Gane for the immunofluorescence tests, and V. Fayet for the preparation of this manuscript. This work was supported by grants from INSERM (National Institute for Health and Medical Research), the Pierre et Marie Curie University, Paris, France, and from CNAM (Caisse Nationale d'Assurance Maladie). References Bloy, C., Blanchard, D., Lambin, P., Goossens, D., Rouger, Ph., Salmon, Ch. and Cartron, J.P. (1987) Human monoclonal antibodies against Rh antigens: partial characterization of the Rh(D) polypeptide from human erythrocytes, submitted. Boylston, A.W., Gardner, B., Anderson, R.L. and HughesJones, N.C. (1980) Production of the human IgM anti-D in
tissue culture by EB-Vims transformed lymphocytes. Scand. J. Immunol. 12, 355. Crawford, D.H., Harrison, J.F., Barlow, M.J., Winger, L. and Huehns, E.R. (1983) Production of human monoclonal antibody to Rhesus D antigen. Lancet 19, 386. Kearney, J.F., Radbruch, A., Liesegang, B. and Rajewski, K., (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody secreting hybrid cell lines. J. Immunol. 123, 1548. Koskimies, S. (1980) Human lymphoblastoid cell line producing antibody against Rh antigen D. Scand. J. Immunol. 11, 73. Kozbor, D. and Roder, J.C. (1981) Requirements for the establishment of high-titered human monoclonal antibodies against tetanus toxoid using the Epstein-Barr virus technique. J. Immunol. 127, 1275. Kozbor, D., Steinitz, M., Klein, G., Koskimies, S. and Makela, O, (1979) Establishment of anti-TNP antibody producing human lymphoid lines by preselection for hapten binding followed by EBV transformation. Scand. J. Immunol. 10, 187. Kozbor, D., Lagarde, H.E. and Roder, J.C. (1982a) Human hybridoma constructed with antigen specific Epstein-Barr virus transformed lines. Proc. Natl. Acad. Sci. U.S.A. 79, 6651. Kozbor, D., Roder, J.C., Chang, M., Steplewski, Z. and Koprowski, H. (1982b) Human anti-tetanus toxoid monoclonal antibody secreted by EBV-transformed human B cells fused with murine myeloma. Hybridoma 1,323. Melamed, M.D., Gordon, J., Ley, S.J., Edgar, D. and HughesJones, N.C. (1985) Senescence of a human lymphoblastoid clone producing anti-Rhesus (D). Eur. J. Immunol. 15,742. Moore, B.P.L (Ed.) (1980) Serological and immunological methods of the Canadian Red Cross Blood Transfusion Service, 8th edn. (Toronto, Ontario). Olsson, L. and Kaplan, H.S. (1980) Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity. Proc. Natl. Acad. Sci. U.S.A., 77, 5429. Reedman, B.M. and Klein, G. (1973) Cellular localization of an Epstein-Barr virus (EBV) associated complement fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11,499. Steinitz, M., Klein, G., Koskimies, S. and Makela, O, (1977) EB-virus induced B lymphocyte cell lines producing specific antibody. Nature 269, 420. Steinitz, M., Izak, G., Cohen, S., Ehrenfeld, M. and Flechner, I. (1980) Continuous production of monoclonal rheumatoid factor by EBV transformed lymphocytes. Nature 287, 443. Vindelov, L. (1977) Flow microfluorometric analysis of nuclear DNA in cells from solid tumors and cell suspensions. Virchows Arch. B. Cell. Pathol. 24, 227. Winger, L., Winger, C., Shastry, P., Russell, A. and Longenecker, M. (1983) Efficient generation in vitro from human peripheral blood cells of monoclonal Epstein-Barr virus transformants producing specific antibody to a variety of antigens without prior deliberate immunization. Proc. Natl. Acad. Sci. U.S.A. 80, 4484. Zurawski, Jr., V.R., Haber, E. and Black, P.H. (1978) Production of antibody to tetanus toxoid by continuous human lymphoblastoid cell lines. Science 199, 1439.