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Human-Human Hybridomas Producing Monoclonal Antibodies Of Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp. 5429-5431, September 1980 Immunology Human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity (somatic human cell hybrids/anti-hapten antibodies) LENNART OLSSON AND HENRY S. KAPLAN Cancer Biology Research Laboratory, Stanford University School of Medicine, Stanford, California 94305 Contributed by Henry S. Kaplan, June 30, 1980 ABSTRACT We report the establishment of human-human 1640/15% FCS/5 ,g of 8-AG per ml. The concentration of hybridomas producing monoclonal antibody of predefined 8-AG was then gradually increased to 20,ug/ml, and viable cells antigenic specificity. The U-266 human myeloma cell line was were cloned in RPMI 1640/15% FCS/20,ug of 8-AG per ml. incubated in the presence of 8-azaguanine, and a rapidly Cultures of the fastest-growing 8-AG-resistant clone were ex- growing, 8-azaguanine-resistant, hypoxanthine/amethop- that were HAT sensitive. This terin/thymidine (HAT) medium-sensitive mutant line, U- panded, after verifying they 266ARI, was selected. These cells were fused with lymphoid mutant cell line, U-266AR1, is routinely maintained in RPMI cells from uninvolved spleens removed at staging laparotomy 1640/15% FCS/5 ,ug of 8-AG per ml. from patients with untreated Hodgkin's disease who had been Human Spleen Lymphoid Cells. Fresh spleen specimens previously sensitized to the chemical allergen 2,4-dinitrochlo- were obtained from untreated patients with Hodgkin's disease robenzene. Hybrid cell cultures growing in HAT medium were undergoing staging laparotomy with splenectomy (9). Only screened for IgG production. Positive cultures were selected and spleens that, on pathological examination, appeared devoid of their supernatants were tested in a solid-phase radioimmuno- involvement by Hodgkin's disease were used. At least 2 weeks assay for reactivity with dinitrophenyl hapten coupled to bovine prior to surgery, such patients are routinely submitted to a serum albumin. Cultures producing specific antibody were subcloned and expanded, and their antibody products were battery of immunologic tests (10), including sensitization and shown to be monoclonal by biosynthetic labeling and sodium later challenge with DNCB. dodecyl sulfate/polyacrylamide gel electrophoresis. A single-cell suspension prepared from the spleen tissue was freed of erythrocytes and granulocytes by Ficoll/Hypaque The production by Kohier and Milstein (1, 2) of mouse "hy- gradient centrifugation (7, 8), and the viable mononuclear cells bridomas" capable of secreting specific monoclonal antibodies were suspended in RPMI 1640 medium. Adherent cells were against predefined antigens has ushered in a new era in ex- removed by incubation of the mononuclear cells in plastic dishes perimental immunology. Essentially all of the problems asso- three times for 20 min each at 37°C and removal of the non- ciated with heteroantisera are circumvented; the clonal selec- adherent cells after each incubation. The lymphocyte-enriched tion and immortality of such hybridoma cell lines assure the mononuclear cell suspensions thus obtained were then fused monoclonality, monospecificity, and permanent availability with the U-266AR1 human myeloma cell line. of their antibody products. At the clinical level, however, the Fusion. For fusion, 2 X 107 myeloma cells and 2 X 107 use of such antibodies would clearly be limited by the fact that lymphoid cells were mixed, washed twice in RPMI 1640 me- are Chimeric have been dium, and then fused in 2.0 ml of 38% (wt/vol) polyethylene they foreign proteins. hybridomas glycol at 37°C. After fusion, the cells were washed twice in generated by fusing mouse myeloma cells with human im- warm (37'C) RPMI 1640 medium, suspended at a concentra- munoglobulin-producing cells (3); however, such hybrids tend tion of 106 cells per ml in RPMI 1640/15% FCS, and seeded in to be unstable due to the progressive loss of human chromo- 0.2-ml samples in microtiter plates with flat-bottom wells somes. Permanent cultures of specific antibody-producing (Falcon no. 3040, Oxnard, CA). Hybrids were selected by in- human B lymphocytes have been obtained by transformation cubation in HAT medium containing 1 MuM hypoxanthine, 63 with Epstein-Barr virus (EBV) (4); this method appears to be nM (+)-amethopterin, and 1.5 MM thymidine. HAT-resistant of limited practical applicability. We describe here a method hybrids grew out within 8-14 days, but incubation in HAT by means of which we have been able reproducibly to obtain medium was continued for at least 3-4 weeks. human monoclonal anti-2,4-dinitrophenyl (DNP) antibodies Antibody Products. Screening for immunoglobulin pro- by fusing 2,4-dinitrochlorobenzene (DNCB)-primed human duction was performed with a solid-phase radioimmunoassay spleen lymphoid cells with human myeloma cells sensitive to using 125I-labeled Staphylococcus aureus protein A as the de- hypoxanthine/amethopterin/thymidine (HAT) medium. In tector. This procedure selectively detects IgG molecules. principle, this method should permit the generation of human Supernatants from cultures positive for staphylococcal pro- monoclonal antibodies against a broad spectrum of predefined tein A-binding IgG production were then tested in a radioim- antigens. munoassay for the presence of antibodies capable of binding specifically with high affinity to DNP conjugated with bovine MATERIALS AND METHODS serum albumin. Several anti-DNP antibody-producing cultures were detected by this procedure. Cells from such wells were Human Myeloma Cell Line. A mutant cell line sensitive to cloned by the limiting dilution procedure, and cultures of the HAT medium (5) was selected from the U-266 human myelo- clone producing the highest level of specific anti-DNP antibody ma cell line described by Nilsson et al. (6). U-266 cells were were expanded. incubated for 1'week in RPMI 1640 medium containing 15% fetal calf serum (FCS) and 8-azaguanine (8-AG) at 20,ug/ml; Abbreviations: DNCB, 2,4-dinitrochlorobenzene; DNP, 2,4-dinitro- dead cells were then removed by Ficoll/Hypaque gradient phenyl; EBV, Epstein-Barr virus; HAT, hypoxanthine/amethop- centrifugation (7, 8) and viable cells were reincubated in RPMI terin/thymidine; FCS, fetal calf serum; 8-AG, 8-azaguanine. 5429 Downloaded by guest on September 29, 2021 5430 Immunology: Olsson and Kaplan Proc. Natl. Acad. Sci. USA 77 (1980) Table 1. Relationship between initial cell concentration and growth of functional hybrids %of wells with % of wells viable cells producing IgG Cells seeded growing at at day per well day 10 after fusion* 21 after fusion 1X109 0, 0, 0 5X105 8, 3, 4 2, 0, 0 2 X 105 54,64,38 29,36,18 .1 1. 1X105 29,32,14 9,11, 6 5X104 32,17, 9 8, 3, 1 I ()() () * Data from three successive experiments with hybridized spleen cells from three patients with Hodgkin's disease. Antibody Characterization. A hybridoma cell clone pro- ducing a high level of anti-DNP antibody was incubated overnight in medium containing [14C]leucine. The immuno- globulins in the supernatant were immunoprecipitated.with rabbit antisera to Fc fragment and human immunoglobulin light chain, and the precipitate was analyzedby sodium dodecyl 2 ()1 '- --- N sulfate (NaDodSO4)/polyacrylamide gel electrophoresis. RESULTS The influence of cell density in the initial wells on the growth of hybrid cells was investigated in a preliminary experiment. Table 1 indicates that 2 X 105 cells per well was the optimal concentration both with respect to hybrid cell growth and IgG production. Screening for anti-DNP antibody production revealed 3, 2, and 0 positive wells, respectively, in experiments with hybrid cells derived from the spleen cells of three successive patients. FIG. 1. NaDodSO4/polyacrylamide gel electrophoresis of Cells from the positive wells were cloned under limiting dilution [14C]leucine-labeled supernatants immunoprecipitated with rabbit conditions. The five specific anti-DNP antibody-producing anti-human Ig. Lane A, parental U-266AR1 myeloma cell line; lane clones thus obtained were then expanded. At optimal hybrid B, H4D11 cloned hybridoma cell line. NL, new light chain. cell concentrations, the amounts of immunoglobulin produced by these clones ranged between 3 and 11 gg/ml per day. The radioimmnnoassay data obtained with these anti-DNP anti- DISCUSSION bodies at the time of initial screening, cloning, and clonal ex- The results reported here demonstrate that it is possible to obtain pansion are presented in Table 2. monoclonal human antibodies by fusion of a HAT-sensitive Production of IgE(X) by the parental U-266 myeloma cell human myeloma cell line (U-266AR1) with antigen-primed line, as reported by Nilsson et al. (6), was confirmed by Na- lymphoid cells. Hybridization of human lymphoid cells with DodSO4/polyacrylamide gel electrophoresis. The selected murine myeloma cells has been reported to result in hybridomas HAT-sensitive mutant of U-266 (the U-266AR1 cell line) was capable of secreting human immunoglobulin (3). However, found to secrete somewhat lower levels of both the eand X chain such hybridoma cells are generally unstable due to progressive products (Fig. 1). Analysis of [14C]leucine-labeled immuno- loss of human chromosomes; this problem seriously limits the globulins produced by hybrid clone H4D11 revealed the pres- use of mouse-human hybridomas for the production of mo- ence of a single additional light (NL) and heavy (,y) chain, noclonal antibodies against predefined antigens. Another ap- confirming the monoclonality of-this antibody product (Fig. proach that has yielded monoclonal antibodies of human origin 1). has been to transform human peripheral blood B cells with EBV and then to select and clone the EBV-transformed cells that Table 2. Specific anti-DNP antibody production by produce antibodies against a defined antigen (4). This technique hybrid human cells requires selection from presensitized human donors of B cells Supernatant No. supernatant Antibody binding,* with the appropriate antigen-binding specificity, a requirement fluid source fluids tested cpm that would ethically exclude the use of many antigens.
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