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Human monoclonal antibodies produced by immortalization with Epstein-Barr virus
38
iramunology today, February 1981
techniques
) Human monoclonal antibodies produced by immortalization with Epstein-Barr virus Michael Steinitz and Eva Klein Department of Hematology, Itadassah University Hospital, Jerusalem, Israel and Department of Tumour Biology, Karolinska Institutet, Stockholm, Sweden Infection with Epstein-Barr virus (EBV) triggers B lymphocytes to proliferate1, 2. B cells which carry the EBV genome can be kept indefinitely in culture while maintaining their marker characteristics, including antibody production. Unless selection pressure is applied, such immortalized cell lines produce polyclonal antibody. Virus can be used to create cell lines producing specific antibody from blood lymphocytes of individuals with high antibody titers against certain antigens. 3 In general, the proportion of antigen-binding cells in the blood does not exceed 0.5% of the lymphocyte population. Because EBV-induced immortalization is a very efficient procedure antibody-producing lines can be established from antigen-binding lymphocytes selected from 10-20 ml blood. Two factors are essential for success: lymphocytes from donors with high antibody titers and the elimination of irrelevant B cells. The procedure involves two steps (1) the enrichment of cells with receptors for the given antigen; and (2) immortalization of these cells by EBV infection. Step 2 is technically easy, step 1 represents the technical limitation which has to be worked out for each system. An alternative strategy, especially in antigen systems in which preselection is impossible, involves the transformation of the total Bcell population with subsequent cloning and testing for antibody-producing cultures. The B cells we have transformed so far were selected by mixing the lymphocyte population with antigencoupled autologous erythrocytes to form rosettes. The denser rosetted lymphocyte sediment when centrifuged through 'Ficoll-isopaque' solution (density 1.077) while the lymphocytes which have not bound erythrocytes remain in the interphase. After separation, the sedimented cells (rosetted lymphocytes and a large excess of antigen-coated erythrocytes) are infected with EBV. 2ml of super© Elsevier~North-Holland Biomedical Press 1981
natant from the EBV-producing cell line B95-8 is added to 0.5 ml of cells and incubated for lh. Thereafter, the cells are centrifuged, the supernatant is removed, 2-3 ml fresh medium is added, and the cells are transferred to tissue-culture flasks. The antigen-coated marker erythrocytes are not removed because they serve a function: their agglutination indicates that an antibody-secreting population has emerged. In addition samples can be removed and the presence of antibodies attached to their surface can be detected, e.g. by immunofluorescence. The emergence of a cell line can be seen after 5-10 days, the first indication being the appearance of lymphoblastic clumps, which usually form a loosely bound complex with the antigen-coated erythrocytes. However, the lymphocyte population usually requires three weeks to reach a sufficient size to produce detectable amounts of antibody in the culture medium. When the lymphocyte population is established, the relevant cell type has to be enriched further by repeating the rosette formation with antigen-coated erythrocytes. To establish an anti-NNP cell line s a culture containing 15% of lymphocytes was reacted with the antigen. From this population the reactive cells were reselected, raising the proportion of antigenbinding cells to 86% and grown. One cell line reactive with the trinitrophenyl (TNP) hapten was established by reselecting three times at three week intervals 6. The proportion of reactive cells was low at first (2%) but after the third selection it was raised to 75%. Other methods can probably also be used for enrichment of the antigen-binding cells. Exposure to fluoresceinated antigen followed by passage in the fluorescence-activated cell sorter is likely to give good enrichment for starting B-cell cultures. However, selection by rosetting is technically simple, does not need large amounts of antigens, and a variety of
immunology today, February 1981
antigens can be coupled to erythrocytes. So far the following lines have been e s t a b l i s h e d : I g M , 1¢ antibodies against N N P (three successes in three trials)3; IgM, 1¢ and ;~ against T N P 6, IgM, • against streptococcal c a r b o h y d r a t e A (two successes in nine trials)7; IgG 1~ anti-tetanus toxoid 8, IgG, ~¢ against Rhesus antigeng; I g M ~ anti-IgG complexed with antigens; i.e. rheumatoid factor (two successes in ten trials) 1°. These results show that cells which secrete various immunoglobulin classes can be immortalized. Like other lymphoblastoid lines, these grow to a density of about 106 cells per ml. Their s u p e r n a t a n t contains 5-20gg a n t i b o d i e s / m l . W e have m a i n t a i n e d some of t h e m for more than a year with no a p p a r e n t change in the nature of their antibody production. T h e y r e m a i n e d diploid. Recently we established a line producing monoclonal r h e u m a t o i d factor from 15 x 106 lymphocytes isolated from a r h e u m a t o i d arthritis patient with a high a n t i b o d y titer against h u m a n or rabbit IgG (1/2048 in the Rose W a a l e r assay) 1°. Preselection was done by rosetting the lymphocytes with autologous erythrocytes coated with h u m a n I g G by the chromic chloride method. After infection with EBV, the growing cells were twice reselected (at three week intervals). T h e antibody they produce binds i m m u n e complexes a n d can be c o m p a r e d to complement in this respect. Because it binds to antibodies complexed with antigens, this I g M is suitable as a reagent for the detection of h u m a n and rabbit antibodies which react
39 with cell-surface antigens. T h e expression of Fc receptors on the target does not h a m p e r its use. In order to obtain lines which manufacture large amounts of immunoglobulin established B-cell lines making monoclonal antibodies can be hybridized with h u m a n myeloma lines. This work was carried out with the support of a Volkswagen Stiftung Grant Az:I/36 120-121. This investigation was in part supported by Grant Number 2 R01 CA 14054-07A1, awarded by the National Cancer Institute, DHEW and by grants from the Swedish Cancer Society.
References 1 Miller, G., and Lipman, M. (1973) Proc. Natl. Acad. Sei. U.S.A., 70, 190-194 2 Einhorn, L., Steinitz, M., Yefenof, E., Ernberg, I., Bak~es, T. and Klein, G. (1978) Cell. Immunol. 35, 43-58 3 Steinitz, M., Klein, G., Koskimies, S. and Mfikel~, O. (1977) Nature (London), 269, 420-422 4 Robinson, J. and Miller, G. (1975) J. Virol. 15, 1065-1072 5 Steinitz, M., Koskimies, S., Klein, G. and M/ikelg_,O. (1979) J. Clin. Lab. Immunol. 2, 1-7 6 Kozbor, D., Steinitz, M., Klein, G., Koskirnies, S. and M/ikel~, O. (1979) Scand. J. Immunol. 10, 187-194 7 Steinitz, M., Sepp~l/i, I., Eichmann, K. and Klein, G. (1979) Immunobiol. 156, 41-47 8 Zurawski, V. R., Jr., Haber, E. and Black, H. P. (1978) Science 199, 1439-1441 9 Koskimies, S. (1979) Scand. J. Immunol. 1I, 73-77 10 Steinitz, M., Izak, G., Cohen, S., Ehrenfeld, M. and Flechner, J. Nature (London), (submitted for publication)
iramunology today, February 1981
techniques
) Human monoclonal antibodies produced by immortalization with Epstein-Barr virus Michael Steinitz and Eva Klein Department of Hematology, Itadassah University Hospital, Jerusalem, Israel and Department of Tumour Biology, Karolinska Institutet, Stockholm, Sweden Infection with Epstein-Barr virus (EBV) triggers B lymphocytes to proliferate1, 2. B cells which carry the EBV genome can be kept indefinitely in culture while maintaining their marker characteristics, including antibody production. Unless selection pressure is applied, such immortalized cell lines produce polyclonal antibody. Virus can be used to create cell lines producing specific antibody from blood lymphocytes of individuals with high antibody titers against certain antigens. 3 In general, the proportion of antigen-binding cells in the blood does not exceed 0.5% of the lymphocyte population. Because EBV-induced immortalization is a very efficient procedure antibody-producing lines can be established from antigen-binding lymphocytes selected from 10-20 ml blood. Two factors are essential for success: lymphocytes from donors with high antibody titers and the elimination of irrelevant B cells. The procedure involves two steps (1) the enrichment of cells with receptors for the given antigen; and (2) immortalization of these cells by EBV infection. Step 2 is technically easy, step 1 represents the technical limitation which has to be worked out for each system. An alternative strategy, especially in antigen systems in which preselection is impossible, involves the transformation of the total Bcell population with subsequent cloning and testing for antibody-producing cultures. The B cells we have transformed so far were selected by mixing the lymphocyte population with antigencoupled autologous erythrocytes to form rosettes. The denser rosetted lymphocyte sediment when centrifuged through 'Ficoll-isopaque' solution (density 1.077) while the lymphocytes which have not bound erythrocytes remain in the interphase. After separation, the sedimented cells (rosetted lymphocytes and a large excess of antigen-coated erythrocytes) are infected with EBV. 2ml of super© Elsevier~North-Holland Biomedical Press 1981
natant from the EBV-producing cell line B95-8 is added to 0.5 ml of cells and incubated for lh. Thereafter, the cells are centrifuged, the supernatant is removed, 2-3 ml fresh medium is added, and the cells are transferred to tissue-culture flasks. The antigen-coated marker erythrocytes are not removed because they serve a function: their agglutination indicates that an antibody-secreting population has emerged. In addition samples can be removed and the presence of antibodies attached to their surface can be detected, e.g. by immunofluorescence. The emergence of a cell line can be seen after 5-10 days, the first indication being the appearance of lymphoblastic clumps, which usually form a loosely bound complex with the antigen-coated erythrocytes. However, the lymphocyte population usually requires three weeks to reach a sufficient size to produce detectable amounts of antibody in the culture medium. When the lymphocyte population is established, the relevant cell type has to be enriched further by repeating the rosette formation with antigen-coated erythrocytes. To establish an anti-NNP cell line s a culture containing 15% of lymphocytes was reacted with the antigen. From this population the reactive cells were reselected, raising the proportion of antigenbinding cells to 86% and grown. One cell line reactive with the trinitrophenyl (TNP) hapten was established by reselecting three times at three week intervals 6. The proportion of reactive cells was low at first (2%) but after the third selection it was raised to 75%. Other methods can probably also be used for enrichment of the antigen-binding cells. Exposure to fluoresceinated antigen followed by passage in the fluorescence-activated cell sorter is likely to give good enrichment for starting B-cell cultures. However, selection by rosetting is technically simple, does not need large amounts of antigens, and a variety of
immunology today, February 1981
antigens can be coupled to erythrocytes. So far the following lines have been e s t a b l i s h e d : I g M , 1¢ antibodies against N N P (three successes in three trials)3; IgM, 1¢ and ;~ against T N P 6, IgM, • against streptococcal c a r b o h y d r a t e A (two successes in nine trials)7; IgG 1~ anti-tetanus toxoid 8, IgG, ~¢ against Rhesus antigeng; I g M ~ anti-IgG complexed with antigens; i.e. rheumatoid factor (two successes in ten trials) 1°. These results show that cells which secrete various immunoglobulin classes can be immortalized. Like other lymphoblastoid lines, these grow to a density of about 106 cells per ml. Their s u p e r n a t a n t contains 5-20gg a n t i b o d i e s / m l . W e have m a i n t a i n e d some of t h e m for more than a year with no a p p a r e n t change in the nature of their antibody production. T h e y r e m a i n e d diploid. Recently we established a line producing monoclonal r h e u m a t o i d factor from 15 x 106 lymphocytes isolated from a r h e u m a t o i d arthritis patient with a high a n t i b o d y titer against h u m a n or rabbit IgG (1/2048 in the Rose W a a l e r assay) 1°. Preselection was done by rosetting the lymphocytes with autologous erythrocytes coated with h u m a n I g G by the chromic chloride method. After infection with EBV, the growing cells were twice reselected (at three week intervals). T h e antibody they produce binds i m m u n e complexes a n d can be c o m p a r e d to complement in this respect. Because it binds to antibodies complexed with antigens, this I g M is suitable as a reagent for the detection of h u m a n and rabbit antibodies which react
39 with cell-surface antigens. T h e expression of Fc receptors on the target does not h a m p e r its use. In order to obtain lines which manufacture large amounts of immunoglobulin established B-cell lines making monoclonal antibodies can be hybridized with h u m a n myeloma lines. This work was carried out with the support of a Volkswagen Stiftung Grant Az:I/36 120-121. This investigation was in part supported by Grant Number 2 R01 CA 14054-07A1, awarded by the National Cancer Institute, DHEW and by grants from the Swedish Cancer Society.
References 1 Miller, G., and Lipman, M. (1973) Proc. Natl. Acad. Sei. U.S.A., 70, 190-194 2 Einhorn, L., Steinitz, M., Yefenof, E., Ernberg, I., Bak~es, T. and Klein, G. (1978) Cell. Immunol. 35, 43-58 3 Steinitz, M., Klein, G., Koskimies, S. and Mfikel~, O. (1977) Nature (London), 269, 420-422 4 Robinson, J. and Miller, G. (1975) J. Virol. 15, 1065-1072 5 Steinitz, M., Koskimies, S., Klein, G. and M/ikelg_,O. (1979) J. Clin. Lab. Immunol. 2, 1-7 6 Kozbor, D., Steinitz, M., Klein, G., Koskirnies, S. and M/ikel~, O. (1979) Scand. J. Immunol. 10, 187-194 7 Steinitz, M., Sepp~l/i, I., Eichmann, K. and Klein, G. (1979) Immunobiol. 156, 41-47 8 Zurawski, V. R., Jr., Haber, E. and Black, H. P. (1978) Science 199, 1439-1441 9 Koskimies, S. (1979) Scand. J. Immunol. 1I, 73-77 10 Steinitz, M., Izak, G., Cohen, S., Ehrenfeld, M. and Flechner, J. Nature (London), (submitted for publication)