Antibody producing human-human hybridomas. I. Technical aspects

Journal of Immunological Methods, 61 (1983) 17- 32 Elsevier

17

Antibody Producing Human-Human Hybridomas. I. Technical Aspects * L. O l s s o n l,**, H. K r o n s t r o m l, A. C a m b o n - D e M o u z o n l, C. H o n s i k 1,2 T. B r o d i n 3 a n d B. J a k o b s e n 4 I Medical Department A, State University Hospital, Copenhagen, Denmark, e Cancer Biology Research Laboratory, Stanford University School of Medicine, Stanford, CA 94305, U.S.A., 3 The Wallenberg Laboratory, University of Lurid, Lund, Sweden and 4 Tissue-Typing Laboratory, State University Hospital Copenhagen, Denmark (Received 23 August 1982, accepted 10 January 1983)

Technical aspects of generation of antibody-secreting human-human hybridomas are evaluated as based on 100 human-human fusions with a human B-lymphoma cell line (RH-L4) or the SKO-007 myeloma cell line as malignant fusion partners, and compared with similar fusion conditions in the mouse hybridoma system. The yield of hybrids was significantly lower when normal peripheral blood lymphocytes were used as fusion partners as compared with spleen lymphocytes, but could be substantially improved by increasing the amount of mitotic active B-lymphocytes by mitogen stimulation of the lymphocytes, preferably in HAT medium, prior to fusion. Furthermore, human hybrids grew slower and had a higher degree of chromosomal instability than usually observed in the mouse hybridoma system. Thus, out of 72 fusions, only 3 stable hybrids with antibody production against a predefined antigen were established. The importance of improved sources of human B-lymphocytes for human-human hybridoma production is discussed and methods of obtaining such improvement suggested. Key words: monoclonal antibody - - human-human hybridoma

Introduction Monoclonal hybridoma antibody technology has already proved to be highly useful in a variety of biological areas (Kennett et al., 1980; H~immerling et al., 1981; * Supported by NIH Grant no. CA-29876, and grants from The Danish Cancer Society, The Danish Medical Research Council, The Carlsberg Foundation, and The Novo Research Foundation. ** To whom correspondence should be addressed. Abbreviations: EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; HAT medium, RPMI 1640 medium with hypoxanthine, aminopterin, and thymidine; LPS, lipopolysaccharide; mPBLs, murine peripheral blood lymphocytes; NP-40, Nonidet P-40; PBLs, peripheral blood lymphocytes; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PWM, pokeweed mitogen; SDS, sodium dodecyl sulfate; SRBC, sheep red blood cells. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.

18 McMichael and Fabre, 1982). Most reports have concerned rodent hybridoma antibodies that are fairly simple to generate although the procedures are tedious and time-consuming. Only a few papers have reported on production of human monoclonal antibodies as produced by human-human hybridomas (Croce et al., 1980; Olsson and Kaplan, 1980; Shoenfeld et al., 1982; Sikora et al., 1982), and none of these reports have dealt with the technical details of the human-human hybridoma system, although in several respects it is more difficult to establish in a reproducible way. We here report on the technical difficulties and improvements of the humanhuman hybridoma system that we have experienced in 100 human-human fusions with emphasis on the procedures that differ from rodent hybridoma technology.

Materials and Methods

Mice Six to 10 weeks old, inbred female B A L B / c mice were used throughout. The mice were obtained from the Panum Institute (University of Copenhagen, Denmark) and kept under conventional conditions. Cell lines All murine fusions were carried out with the non-producer X63-Ag.8.6.5.3. mouse myeloma line (Kearney et al., 1979). Two human cell lines were used. The majority of the human-human fusions was carried out by using a HAT-sensitive, EBV-negative human B-lymphoma line (RH-L4) that produces, but does not secrete, low amounts of 7-heavy chain and x-light chain; some experiments were done with a mycoplasma-cleaned variant of the SKO-007 line secreting low amounts of e-heavy chain and rather high amounts of ~,-light chain (Olsson and Kaplan, 1980). The human cell lines were maintained in RPMI 1640 medium with 15% fetal calf serum supplemented with 0.3% L-glutamine (in addition to L-glutamine in the RPMI 1640 medium) and 40/~g/ml 8-azaguanine. Both human lines were normally seeded at a concentration of 105 cells/ml and used for fusion 3-5 days after seeding. The RH-L4 line has a doubling time of 30 h and the SKO-007 variant doubles in 55 h. Lymphoid cell fusion partners Mouse-mouse fusions were done both with mononuclear spleen cells and mononuclear cells from peripheral blood (mPBLs). Both preparations were depleted of erythrocytes and granulocytes on a Ficoll-Hypaque gradient (density 1.09 g/ml), w~shed twice in cold RPMI 1640 medium and then processed in the fusion procedure. All the human fusions were done with human lymphoid cells from peripheral blood. In some experiments, mice were immunized with sheep red blood ceils (SRBC) by i.p. injection of 5 x 10 7 SRBC day 0 and day 14, and spleen cells a,:a mPBLs harvested 4 days later. In all cases, the mononuclear cells were isolated on Ficoll-Hypaque (density 1.07 g/ml; Nyegaard, Oslo), washed twice in RPMI 1640 medium and then fused with human myeloma/lymphoma cells.

19

Fusion procedures and HA T selection All fusions were carried out in 37% w / v polyethylene glycol (PEG), MW 1000 (Baker, U.S.A.) at room temperature. Lymphocyte:myeloma or lymphoma ratios were 1 : 1 in both human-human and mouse-mouse fusions. The cells were seeded at a concentration of 2 × 105 cells/well in 96-well microtiter plates (Nunc, Denmark) in H T medium with 15% FCS. Hybrid selection in HAT medium was started 24-48 h after fusion by exchanging most of the H T medium with HAT medium. Our myeloma and lymphoma cell lines are tested for H A T sensitivity every 6-10 weeks and twice the aminopterin concentration that kills all myeloma/lymphoma cells is used in the H A T medium. Typically, RH-L4 cells die within 10 days in HAT medium with an aminopterin concentration of 10 -8 M, and the H A T medium thus c o n t a i n s 10 - 4 M hypoxanthine, 2 x 10 -8 aminopterine, and 2 x 10 - 6 M thymidine (all Sigma Chemical Co., U.S.A. ) in RPMI 1640 culture medium with 15% non-inactivated FCS. Half of the H A T medium is renewed every 3 days in the first 2 weeks after fusion, and the HAT medium is then gradually replaced by H T medium within 4 - 6 days. In the mouse system, the hypoxanthine and thymidine concentrations were as above, but the aminopterin concentration was 2 x 10 -7 M. Feeder cells and cloning The importance of optimal feeder cell sources for human hybridoma growth has been described in detail elsewhere (Brodin et al., in press). Briefly, monocytes from fresh 'buffy coats' are isolated either by their ability to adhere to the bottom of wells in 96-well plates or on gelatine columns. About 10-50 x 103 monocytes are seeded per well and addition of a similar number of feeder cells was occasionally necessary every 10-14 days during the initial culture period, as the quality of the monocytes as feeder cells varied between buffy coats. Human and murine thymocytes could also be used as feeders (in a concentration of 5 x 105 cells/well), whereas murine spleen or murine peritoneal macrophages to some extent phagocytized the human hybridomas. Mouse-mouse hybrids were fed with mouse thymocytes (5 x 105 cells/well) throughout. Cloning of hybrids was done by limiting dilution with a minimum of 1 cell/well. Mitogen stimulation Lymphoid cells were in some experiments mitogen-stimulated with pokeweed mitogen (PWM) (final dilution 2.5 /xg/ml; Gibco Laboratories, U.S.A.) to test the fusion ability of PWM-stimulated cells at various times after the mitogen st?mulation. The degree of DNA activity was determined by incubation of the cells in [3H]thymidine ([3H]TdR; 10/.~Ci/ml, specific activity 6.0 C i / m M ; NEN, U.S.A.) for 18 h, and the [3H]TdR content in 7% trichloroacetic acid insoluble cell material determined by liquid scintillation. Enzyme-linked immunosorbent assays (ELISA) Hybridoma culture supernatants were initially tested for antibody in an ordinary ELISA. Normally, this requires harvesting of supernatant fluids from the cultures with a possibility of disturbing the cultures (and contaminating them) so that a

20

Fig. 1. Plastic device for testing hybridoma tissue culture supernatants for antibody. The device is a plastic lid with 96 sticks that fit into wells of a 96-well microtiter plate. The sticks are incubated in hybridoma tissue culture supernatants for 1-2 h and then further processed in the ELISA. The sticks may be precoated with antigen or antibody.

r e p e t i t i o n of the assay c a n n o t b e p e r f o r m e d until 3 - 6 d a y s later. W e (L. Olsson a n d N u n c , Roskilde) therefore d e v e l o p e d a device (Fig. 1; N u n c - T S P , Roskilde) that allows testing of h y b r i d o m a cultures for a n t i b o d y w i t h o u t s u p e r n a t a n t harvest a n d with the p o s s i b i l i t y of r e p e a t e d testing on consecutive days. The device is a 96-well p l a s t i c lid that has sticks fitting into the wells. T h e sticks are m a d e of plastic with high a n t i b o d y - b i n d i n g capacity, a n d d o not touch the b o t t o m of the well; they can b e inserted into wells that c o n t a i n up to 150 /~1 s u p e r n a t a n t fluid. T h e sticks are i n c u b a t e d for 2 h at 20°C in the s u p e r n a t a n t s , washed twice, then i n c u b a t e d with the s e c o n d reagent solution (e.g., p h o s p h a t a s e - c o n j u g a t e d r a b b i t a n t i - h u m a n Ig or r a b b i t a n t i - m o u s e Ig) for 1 h at 20°C, w a s h e d 5 times in 1% BSA-PBS, a n d transferred to 96-well plates c o n t a i n i n g the staining substrate. The plates are r e a d 15 min a n d 45 m i n after transfer of the sticks into the s u b s t r a t e on an a u t o m a t i c m u l t i s c a n n e r

21

(Multiscan; Flow Lab., U.S.A.). The device has a detection level of 10-50 ng antibody per ml supernatant and has increased 8-10 times our capacity to screen initial hybridoma culture supernatants for antibody. Furthermore, the system permits direct specificity testing of the antibody in cases, where antigen can be coupled to the sticks. Cell-binding ELISA was performed by conventional method (Suter et al., 1980). The cells were seeded in a concentration of 5 × l 0 4 per well in wells of microtiter plates coated with poly-L-lysin and fixed in 0.1% glutaraldehyde. Fifty microliters of supernatant were diluted 1 : 1 with PBS, pipetted into the wells, and the ELISA test performed as described above.

Antibody analyses Hybridoma produced antibodies were labeled with [35S]methionine by incubation overnight of 106 hybridoma cells in 1 ml methionine-free RPMI 1640 medium supplemented with 50 ktCi [35S]methionine (NEN, U.S.A.). The supernatants from such cultures were harvested and Ig products precipitated by the following procedure: 80/~1 of goat anti-Hulg (Dako, Denmark) at a dilution of 1 : 100 was added to 0.25 ml [35S]methionine-labeled hybridoma supernatant and incubated on ice overnight. Eighty microliters of a rabbit anti-goat Ig was then added to a final dilution of 1:50, and after 6 h incubation on ice, 50 /~1 prewashed protein A-Sepharose (Pharmacia, Sweden) was added. The suspension was incubated for 1 h at 4°C under constant stirring, centrifuged, and the pellet washed and used for further sodium dodecylsulfate (NaDodSO4)/polyacrylamide (SDS) gel electrophoresis. SDS analyses were performed as described elsewhere (Cowan et al., 1974; O'Farrell et al., 1977). The immunoglobulin production rate of human hybridomas was assayed in an inhibition assay. Wells in 96-well plates were coated with human Ig (IgA + IgM + IgG) at a concentration of 1 /~g/well. The wells were washed twice and unblocked plastic sites coated by incubation in PBS with 10% FCS for 30 min at room temperature. Twenty-five microliters of test supernatant mixed with 25 /~1 rabbit anti-human Ig (conjugated with phosphatase and diluted 1 : 500 in PBS) was added to the wells and incubated for 45 min at 37°C. The plates were then washed twice in PBS, 150/~1 substrate solution for ELISA testing added and the plates read after 15 and 45 min incubation at room temperature. The titration standard c u r v e w a s obtained by using normal human serum in dilutions 1 : 102 to 1 : 10 6. Fluorescence-activated cell sorter analyses. A number of analyses were performed on a FACS IV machine (Becton-Dickinson, Sunnyvale, CA). Two-step fluorescence staining of cells for detection of cell surface-directed antibodies in culture supernatants was done by incubation of 10 6 cells in 100/~1 supernatant for 1 h on ice, followed by 3 washings and then incubation on ice for 30 min with FITC-conjugated rabbit anti-human Ig or rabbit anti-mouse Ig (Dako, Denmark) on ice. After 3 more washes the cells were resuspended in PBS, and analyzed. Both sorting and analysis procedures were done with a 70/~m nozzle tip at a rate of 1500-3000 cells per sec. Microcytotoxicity assay. The test was carried out in a conventional tissue typing system as described in detail elsewhere (Terasaki et al., 1972).

22

DNA analyses. Banded karyograms were done by conventional procedures. The D N A content of nuclei of parental cells and hybridoma cells was measured on propidium iodine stained isolated nuclei (Vindel~v, 1977). Briefly, the cytoplasm was removed with 0.1% w / v NP-40 detergent solution, the nuclei stained in a 0.04% w / v propidium iodine solution, and the DNA content of the nuclei measured on the FACS IV machine.

Results

Initial hybridoma yield Table I gives the results obtained in 100 human-human fusion experiments of which 53 were done with the RH-L4 line and 47 with the SKO-007 myeloma variant. The frequency of fusions that resulted in hybrids varied widely from 7% to 70% in the various experimental groups and so did the number of wells with hybrids in those fusions that resulted in hybrids (4-56%). The number of fusions resulting in hybrids was slightly higher with the RH-L4 line (39%) then with the SKO-007 line (19%), and the number of wells with I g G / I g M production was significantly higher with RH-L4 fusions than SKO-007 fusions (51% versus 35%; P < 0.01 with x2-test). Among the 100 fusions, only 21 resulted in antibody producing hybrids and in these 21 fusions the average number of wells with Ig-producing hybrids was 11%. Only 15 wells contained antibody-producing hybrids with predefined specificity in 72 fusions (about 18,000 wells). The number of specific hybrids, and hybrids with specific Ig-production, was thus very low as compared with conventional mouse-mouse fusions with optimally antigen-primed spleen cells (Table II). However, the relevant comparison between the 2 systems is to compare the hybridoma yield, when mouse PBLs are fused with X63 Ag/8.6.5.3 and human PBLs with RH-L4 or SKO-007 cells. Table II thus also demonstrates the very low yield of hybrids in the groups using mouse PBLs. It should be noted that the fusions with mPBLs from mice optimally primed with SRBC resulted in only two hybrids producing antibody against SRBC.

Cloning and stability of human hybridomas The 100 fusions recorded in Table I resulted in all in 21 wells with hybrids producing antibody against antigens with known specificity (Table II). Only 3 of these 21 cultures were, however, successfully cloned and re-expanded, as almost half of the cultures died out during cloning, a few lost Ig production, and one culture died out during attempts to expand the culture in flasks. Thus, out of 100 fusions we only obtained 3 hybrid cultures producing antibody of interest.

Pokeweed-mitogen stimulation of human PBI.s prior to fusion PEG mediates only fusion of the cell membranes of the myeloma cell and the B-lymphocyte, whereas fusion of the karyons occurs spontaneously when the 2 nuclei synchronously enter mitosis. However, human PBLs are normally not in active cell cycle, and fusions of such cells with a human myeloma/lymphoma cell

3 (1) 3 (1) 4 (3) 10 (10) 8 (8)

RH-L4 SKO-007

(14) (14) (22) (22)

(14%) (7%) (18%) (9%)

7 (70%) 5 (50%)

1 (33%) 1 (33%) 2 (50%)

2 l 4 2

No. of fusions with visible hybrid growth 30 days after fusion

41; 25; 14 37, 21;

8; 18 12; 9 6; 28 14 9;

54; 4; 9

12; 6; 32; 9;

64

19

1; 56;

21

% Wells in microtiter plates with visible hybrid growth 30 days after fusion

34; 2; 1; 18; 20; 0; 6 14; 14; 0; 16; 41

21 6 1; 21

0; 15 3 4; 0; 30; 4 2; 5

% Wells with IgG of IgM in supernanatants

0/200; 0/200; 0/200; 0/300 e; 0/260; 0/200; 0/260 0/300; 0/260; 0/280; 0/300; 0/300

4/300 c 0/300 0/200; 11/300 d

ND a ND 0/200; 0/200; 6/200 b 0/200; 0/200

No. of wells with Ig specific for predefined antigen/total no. of wells

a Not determined. b Screened in cell-binding ELISA test against the patients's own myeloid leukemia cells and AML-cells from 3 other patients. c Tested in ELISA with purified tetanus toxoid as antigen. d Tested in cell-binding ELISA with erythrocytes presenting relevant antigens. c Tested in microlymphocytotoxicity against a panel of different HLA-types.

Patients with high titer of anti-D blood group or B-antibody Patients with high titer anti-HLADR antibodies

Tetanus toxoid immunized person

14 14 22 22

No. of fusions (no. patients)

RH-IA SKO-007 RH-L4

RH-L4 SKO-007 RH-L4 SKO-007

Healthy normal individuals

Acute myeloid leukemia patients

Malignant fusion partner

Source of PBLs

YIELD OF H U M A N - H U M A N HYBRIDOMAS A F T E R FUSION OF PERIPHERAL BLOOD LYMPHOCYTES (PBLs) WITH A H U M A N MYELOMA (SKO-007) OR LYMPHOMA (RH-L4) CELL LINE. THE FUSION WAS DONE ON THE SAME DAY AS THE BLOOD SAMPLE WAS T A K E N F R O M THE PATIENT

TABLE I

24 T A B L E II Y I E L D OF M O U S E - M O U S E HYBR1DOMAS A F T E R F U S I O N OF T H E M U R I N E X63.Ag.8.6.5.3 W I T H M O U S E L Y M P H O I D CELLS F R O M SPLEEN OR P E R I P H E R A L BLOOD (PBLs) a Source of lymphoid cells

SRBC priming

No. of experiments

% Wells with hybrid growth 30 days after fusion

% Wells with I g G / I g M in supernatant

No. of wells with antibody against S B R C / T o t a l no. of cells

Spleen

None +

4 4

18; 29; 4; 38; 64; 52; 74; 68

8; 21; 0; 22 52; 40; 61; 46

0/300; 0/300; 0/300; 0 / 3 0 0 9/300; 7/300; 11/300; 5 / 3 0 0

PBLs

None +

4 4

0; 4; 0; 1 8; 10; 0; 2

0; 1; 0; 0 4; 6; 0; 1

0/200; 0/200; 0/200; 0 / 2 0 0 0/180; 0/120; 1/180; 1/180

a Mouse lymphocytes were harvested 4 days after SRBC priming.

T A B L E III C L O N I N G E F F I C I E N C Y BY L I M I T I N G D I L U T I O N A N D STABILITY OF H U M A N - H U M A N HYBRIDS No. of wells to be cloned

No. of wells lost in the cloning procedure

Cloning efficiency in % of the 10 successfully cloned hybrid cultures

No. of hybrid cultures that lost I g G / I g M production during cloning

No. of hybrid cultures that after cloning was expanded in flasks

No. of cloned hybrid cultures producing lgG/lgM 80 days after fusion

21

11 (52%)

24 (3-42)

6 (29%)

4 (19%)

3 (14%)

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~

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2 3 4 5 6 7 Days of PWM stimulation

8

Fig. 2. Effect of pokeweed mitogen stimulation of h u m a n PBLs on ability to fuse with a h u m a n B-lymphoma cell line (RH-L4). The yield of hybridomas is significantly ( P < 0.01; x2-test) increased, when 5 - 7 days PWM-stimulated lymphocytes are used for fusion. The results are the means of 3 experiments.

25

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Fig. 3. Effect of HAT medium on PWM stimulation of human PBLs. PWM was added at day 0. The cells were incubated in RPMI 1640 with 10% FCS or RPMI 1640 with 10% FCS, 1 0 - 4 M hypoxanthine, 2 x 10-8 M aminopterine, and 2 x 10-6 M thymidine. The d&y before assessing DNA synthesis activity, the cells were washed twice in RPMI 1640, incubated overnight in RPMI 1640 plus 10% FCS supplemented with [ 3H]thymidine and the incorporation of [3 H]TdR subsequently determined.

line will therefore not result in functional mononuclear hybridoma cells. PBLs were therefore stimulated with pokeweed mitogen (PWM) prior to fusion and Fig. 2 shows the impact of PWM stimulation on the yield of hybridomas. Five to 7 days after PWM stimulation was the optimal time for fusion of PWM stimulated PBLs with the parental fusion partner. The PWM stimulation is done in HAT medium in order to adapt the lymphocytes to the DNA synthesis rescue pathway that the hybrids use to overcome the H A T blockage. This resulted in a 60% decrease in the

TABLE IV YIELD OF H U M A N - H U M A N HYBRIDOMAS AFTER FUSION OF 6 DAY PWM-STIMULATED H U M A N PBLs WITH THE RH-L4 CELL LINE. Source of PBLs

No. of fusions (No. of patients)

No. of fusions with visible hybrid growth 30 days after fusion

(Mean and range) % Wells in microtiter plates with visible hybrid growth 30 days after fusion

(Mean and range) % Wells with IgG or IgM in supernatants 30 days after fusion

No. of wells cloned

No. of cloned hybrid cultures producing I g G / IgM 80 days after fusion

Healthy normal individuals

40 (38)

14 (35%)

31 (4-52)

17 (10-34) 11; 9; 22; 22; 14; 19; 36; 20

30

8 (27%)

26 O.D.

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E'--~

NH

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X~ ~'- NL

A

B

'*--NL

C

D

Fig. 5. SDS analysis of [35S]methionine-labeled Ig products. A, SKO-007 human myeloma cell line with its ~-heavy chain and X-light chain; B, H23RH-12 hybridoma generated by fusion of SKO-007 with human PBLs; NH, new heavy chain and NL, new light chain; C, RH-L4 human B-lymphoma cell line; D, H62-AML-18 hybridoma generated by fusion of RH-L4 with human PBLs with secretion of the parental x-light chain and a new heavy chain (NH) and new light chain (NL).

27 TABLE V IMMUNOGLOBULIN CHAIN TYPES PRODUCED BY HUMAN-HUMAN HYBRIDOMAS 40 DAYS AFTER FUSION Source of B-lymphocyte fusion partner

Malignant fusion partner

Total no. of hybrids

Ig-type y

/t

c

)k

K

PBLs

SKO-007 RH-L4

20 20

9 20

4 5

20 0

20 3

11 20

PWM-PBLs

RH-L4

30

30

7

0

4

30

total amount of lymphocyte that can be harvested (Fig. 3) at the optimal time for fusion with the myeloma/lymphoma line. Forty fusions of PWM-PBLs with RH-L4 resulted in 14 with hybrid growth, which is significantly higher (P < 0.01; X2-test) than with fusions of PBLs with RH-L4 cells, or with fusions with PBLs stimulated in PWM in RPMI 1640 medium without HAT (data not shown). The number of

A

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,

)

[

B

C D Fig. 6. DNA analysis of hybridoma cultures in relation to days after fusion. (A), normal human PBLs channel 38-53, Gl-peak channel 45; B, 5 days PWM-stimulated human PBLs, channel 41-104, GI peak in channel 46, G2-peak in channel 104; C, RH-L4 human B-lymphoma cell line channel 44-112, Gl-peak in channel 56, G2-peak in channel 107; D, cloned H62-AML-18 hybridoma 90 days after fusion with RH-L4 cells, channel 51-133, Gl-peak in channel 61, G2-peak in channel 115.

28 TABLE VI FACS ANALYSIS OF DNA C O N T E N T OF N U C L E I FROM N O R M A L PBLs , THE RH-L4 H U M A N B-LYMPHOMA LINE, A N D TWO HYBRIDS OBTAINED BY F U S I O N OF PBLs WITH RH-L4 Cell types

Gl-peak marker position

G2-peak marker position

Channel interval that contains 99% of the cells

% Cells in the channel number interval 125255

PBLs PWM-stimulated PBLs RH-L4 lymphoma cells H u m a n hybridoma culture (Hu-HyF 18) H u m a n hybridoma culture (Hu-HyM37)

47 47 58

No cells 92 111

44- 56 44 96 51-126

0 0 0.4

66

123

55-242

6.9

62

125

54-232

5.4

Ig-secreting hybrids tended also to be higher in the fusions using PWM-PBLs, although not significantly (Table IV).

Antibody analysis Screening of hybridoma cultures was done by ELISA testing. Fig. 4 demonstrates typical values obtained in fusions with lymphocytes from AML patients. It is seen that the cell-binding ELISA assay gives significantly higher values ( P < 0 . 0 1 ; Rang-sum test) than the values for Ig production. It should also be noted that 2 of the supernatants positive in the cell-binding ELISA were negative on analysis for Ig production. Table V shows the types of immunoglobulins that were secreted in a Chromosome counts in a non-cloned human hybridoma culture 1month a tter fusion 20

16

E

12

.O

E

8

4

31- 36-41- 46-51- 56-61- 66-71- 76- 81- 86-91- 9635 4 0 45 50 5 5 60 65 70 75 8 0 85 90 95 100 N ° chromosomes per cell

Fig. 7. Chromosome analysis of a non-cloned human hybridoma culture 30 days after fusion. The mitotic arrest was obtained by incubation of the cells for 2 h in colcemid supplemented medium and the cells were then processed as described by Hsu (1974). 150 consecutive mitotic figures were counted.

29 number of randomly selected hybrids. The RH-L4 cells produce but do not secrete 3', K. However, the hybrids of RH-L4 cells and lymphocytes did secrete "y, x, but also in some cases/~, ~. A few of the hybrids (4) secreted only a new light chain. The amount of secreted Ig was between 0.5-9.0 ~tg per ml 106 hybrids per 24 h. Fig. 5 shows SDS electrophoresis of [35S]methionine-labeled secretion Ig products of the SKO-007 malignant parental fusion partners and a few hybrids.

DNA analysis The hybrids appeared with a triploid to tetraploid chromosome content 3 weeks after fusion, but then underwent very rapid D N A loss. Forty days after fusion, most of the cells had a diploid to triploid content. Stable hybrids were found to have a D N A content of 2N-3N in most cases (Table VI and Fig. 6). This concurs with a chromosome analysis on a non-cloned hybridoma culture 1 month after fusion (Fig. 7), and with a karyogram analysis of a cloned hybridoma culture producing antibody against AML cells (Fig. 8).

Discussion

Monoclonal antibodies have great promise for application in a number of biomedical areas, including diagnosis and therapy of human diseases. It is to be expected that human monoclonal antibodies are better tolerated upon injection into human beings than similar mouse antibodies. A few groups have reported successful human-human hybridoma production (e.g. Croce et al., 1980; Olsson and Kaplan, 1980), but many laboratories have been unsuccessful with such hybrids, and this raises questions about the practical usefulness of the human-human hybridoma system in its present form. The rodent hybridoma system is highly dependent on optimal antigen-primed B-lymphocytes (Oi et al., 1978; Goding, 1980), This can easily be obtained in laboratory animals, but similar in vivo primed human B-lymphocytes are practically impossible to obtain in human beings. The present paper describes the results of 100 human-human fusions and underlines the importance of access to B-lymphocytes at a certain stage of differentiation that at present we do not know details of. It also suggests that too much attention may have been paid to the malignant fusion partner. Out of 72 fusions and more than 18,000 wells (each starting with 2 × 105 cells; i.e., a total of more than 3.6 × 109 cells), only 3 cloned hybrid cultures of interest were obtained. This is drastically lower than the rates obtained for mousemouse fusions with antigen-primed spleen cells. However, our results also show that the correct comparison between the murine and human systems (mouse PBLs fused with a mouse myeloma line versus human PBLs fused with human cells) gives similar poor yields of hybrids in the mouse-mouse hybridoma system. One of the reasons of this is probably that PBLs are not in active mitotic cell cycle. PEG mediates fusion of cell membranes of PBLs and myeloma cells, but not of karyons. We therefore triggered a high number of PBLs into mitosis by PWM and our results demonstrate an improved yield of hybrids. This concurs with Pasley and

30 Roozen's (1981) recent demonstration of increased hybridoma yield by LPS stimulation of mouse spleen cells prior to fusion and also with the observation of increased hybridoma yield by boosting with antigen in vivo or in vitro (Fox et al., 1981). The antibody analysis was done in a rapid screening ELISA assay. However, some of the supernatants positive for a given antigen were not detected in the ELISA assay for Ig production, underlining the importance of using several screening

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.

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Fig. 8. Karyogram analyses of: A, RH-L4 B-lymphoma HAT-sensitive cells; 50, xy, - I , + t ( l ; ?), + t ( l ; ?), 5 p + , 6 q + , +7, 8 q + , - 2 2 , + 4 mar. B. cloned human-human hybridoma with antibody production 8 weeks after fusion; 75, xy, + t (1 ; ?), + t(l ; ?), + 2, + 2, + 4, 5p + , 6q - , + 7, + 7, + 7, 8q + , - 1 0 , + 11, + 12, + X , + 13, + 15, + 19, +20, + 14 mar. C, same culture 8 months after fusion still Pr0ducing antibody; 55, xy, - 1 , + t (1; ?), +t(I; ?), +2, - 3 , +4, 5 p + , 6 q - , +7, +7, 8 q + , - 9 , +X, - 22, + 6 mar.

assays. In general, we use ELISAs both for Ig production and antigen-specific antibody production as initial assays, supplemented for cell-surface antigens with microcytotoxicity tests and cell sorter analysis. With these 4 types of test we require 2 of the 4 to be positive in order to persist with a given hybrid culture. Antibody production was very variable, but was in a range comparable with that of the mouse system. The stability of the hybrids was a significant consideration in the human system, as a large number were lost during attempts to expand the cultures. The D N A analysis and chromosome studies indicate that this instability may be due to extensive chromosome loss in the hybrids in the first 3-8 weeks after fusion. Repeated cloning of the hybrids was thus necessary to isolate a stable Ig-producing hybridoma. We probably lost a high amount of Ig-producing human-human hybrids due to overgrowth of non-producers, to a greater extent than we are used to in the mouse hybridoma system in our laboratory. Mitogen stimulation of PBLs in H A T medium as well as optimal feeder cell conditions greatly improved the hybridoma yield, although it remained significantly lower as compared with the mouse system. We think that this difference is mainly due to the suboptimal source of antigen-primed cells, as indicated by studies on the frequency of specific antigen primed cells (Stevens et al., 1979), and to some extent to a suboptimal malignant fusion partner. It seems therefore of crucial importance

32

for the human-human hybridoma system that in vitro antigen-priming systems are developed in line with those already published to promote a primary immune response (Hoffman, 1980; Volkman et al., 1981) or a recall response (Lane et al., 1982). The recent development of long-term B-lymphocyte cultures (Howard et al., 1981; Sredni et al., 1981) thus opens up further the possibility that long-term cultures of normal B-cell cultures with specific antibody production can be established, and may in time replace hybridoma technology in the production of human monoclonal antibodies.

References Brodin, T., L. Olsson and H.O. Sjt~gren, J. Immunol. Methods, in press. Cowan, N.J., D.S. Sechner and C. Milstein, 1974, J. Mol. Biol. 90, 691. Croce, C.A., A. Linnenbach, W. Hall, Z. Steplewski, and H. Korpowski, 1980, Nature (London) 288, 488. Fox, P.C., E.H. Berenstein and R.P. Siragawian, 1981, Eur J. Immunol. 11,431. Godin, J.W., 1980, J. Immunol. Methods 39, 285. H~immerling, G.J., U. H~mmerling and J.F. Kearney (eds.), 1981), Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, Amsterdam) p. 1. Hoffman, M.K., 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 1139. Howard, M., S. Kessler, T. Chused and W.E. Paul, 1981, Proc. Natl. Acad. Sci U.S.A. 78, 5788. Hsu, T.C., in: Tissue Culture. Methods and Applications, eds. P.F. Kruse and M.K. Patterson (Academic Press, New York) p. 764. Kearney, J.F., A, Radbruch, B. Liezengang and K. Rajewsky, 1979, J. Immunol. 123, 1548. Kennett, R.H., T.J. McKearn and K.B. Bechtol (eds.) 1980, Monoclonal Antibodies (Plenum Publishing Corporation, New York) p. 1. Lane, H.C., J.H. Shelhamer, H.S. Moztowski and A.S. Fauci, 1982, J. Exp. Med. 155, 333. McMichael, A. and J. Fabre (eds.), 1982, Monoclonal Antibodies in Clinical Medicine (Academic Press, New York) p. 1. O'Farrell, P.Z., H.M. Goodman and P.H. O'Farrell, 1977, Cell 12, 1133. Oi, V.T., P.P. Jones, J.W. Goding, L.A. Herzenberg and L.A. Herzenberg, 1978, Curr. Top. Microbiol. Immunol. 81, 115. Olsson, L. and H.S. Kaplan, 1980, Proc. Natl. Acad. Sci. U.S.A. 77, 5429. Pasley, J.W. and K.J. Roozen, 1981, in: Monoclonal Antibodies and T-Cell Hybridomas, eds. G.J. H~mmerling, U. H~immerling and J.F. Kearney (Elsevier, Amsterdam) p. 551. Shoenfeld, Y., S.C. Hsu-Lin, J.E. Gabriels, L.E. Silberstein, B.C. Furie, B. Furie, B.D. Stoller and R.S. Schwartz, 1982, J. Clin. Invest. 70, 205. Sikora, K., T. Anderson, J. Phillips and J.V. Watson, 1982, Lancet i, l 1. Sredni, B., D.G. Sieckmann, S. Kumangai, S. House, I. Green and W.E. Paul, 1981, J. Exp. Med. 154, 1500.

Stevens, R.H., E. Macy, C. Morrow and A. Saxon, 1979, J. Immunol. 122, 2498. Surer, L., J. Brtigger and C. Sorg, 1980, J. Immunol. Methods 39, 407. Terasaki, P.J., B. McCurdy and J.B. McClelan, 1972, in: Manual of Tissue Typing Techniques, eds. J.G. Ray, R.C. Scott, D.B. Hare, C.E. Harris and D.E. Kayhoe (NIH, Bethesda, MD) p. 50. Vindel~v, L., 1977, Virchows Arch. B. Cell Pathol. 24, 227. Volkman, D.J, C.L. Lane and A.S. Fauci, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 2528.