Recent improvements in the production of antibody-secreting hybridoma cells

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Recent improvements in the production of a nti body-secreti n g

hybridoma cells GarryA. Neil and Howard B. Urnovitz The field of cell fusion technology has exploded over the past ten years, revolutionizing biology and medicine. Today, monoclonal antibodies specific for almost any antigen can be generated, setting the stage for m a n y different applications. Continued research that focuses on refining the technology and developing innovative techniques will enable the field of cell fusion technology to reach its full potential. Since 1975, w h e n K6hler and Milstein showed that mouse myeloma cells could be fused to B lymphocytes from immunized mice resulting in continuously growing, specific monoclonal antibody-secreting (mAbsecreting) somatic cell hybrids or 'hybridoma' cells 1, the technique has become widely adopted. Today hundreds of research and diagnostic procedures are based upon this technology, and mAbs are n o w being introduced as therapeutic reagents in clinical trials 2. A measure of the impact that the technology currently enjoys is the 300 to 400 artic]es containing the words 'hybridoma' or 'monoclonal antibody' in their titles or abstracts published each month 3. Using conventional and generally available techniques, researchers can n o w generate and characterize homogeneous antibodies specific for almost any antigen, evaluate their usefulness in given procedures, and efficiently generate large quantities of reagent-grade mAbs for a given application. Garry Nell is at the Department of Medicine, University of Iowa College of Medicine, Iowa City, 1,4 52242. Howard Urnovitz is at Calypte Biomedical Company Inc., 1440 Fourth Street, Berkeley, CA 94710, USA. 1988,

Elsevier Publications, Cambridge

Yet the technology is still developing; there are inconsistencies in some techniques and some unsolved practical problems. For example, it remains difficult to generate mAbs of high affinity and adequate specificity to very weak immunogens or antigens that are available in only minute quantities (as is often the case for cancer-associated antigens). Furthermore, it is not possible to control the class or subclass of resulting antibodies, a characteristic that often determines their biological activity and therefore their usefulness. In addition, existing immunization protocols severely limit the generation of human mAbs (which may ultimately prove to be the most useful antibodies for in-vivo diagnostic and therapeutic purposes). Lastly, production of useful reagents often involves labor- and time-intensive procedures that greatly lengthen their production time and, ultimately, their cost.

Standard methods Most laboratories engaged in the production of mAbs currently use a variation (for example Ref. 4) of the polyethylene glycol (PEG) fusion technique 5, although the old maxim - that there are as many fusion protocols as there are immunologists

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- appears to have some validity. In brief, experimental animals (usually mice or rats, although hamsters are now becoming widely used for specific purposes) 6 are immunized by injecting them with soluble antigen or cells and an immunoadjuvant (e.g. Freund's adjuvant or a suspension of Bordetella pertussis). Three or four weeks later, the animals receive a booster injection of the same antigen without the adjuvant. Three to five days after this second injection, their spleens are excised. The spleen cells are mixed with an appropriate 'fusion partner' which is usually a non-secreting hybridoma or myeloma cell, often SP2/0 (Ref. 7). The cell suspension is then briefly exposed to a solution of PEG to induce fusion by reversibly disrupting the membranes. The PEG is then diluted and the cells washed in growth medium. Then they are cultured in growth medium containing hypoxanthine, aminopterin and thymidine (HAT), which permits the survival of only those cells expressing hypoxanthine guanine phosphoribosyl transferase, i.e. the hybridoma cells. After between four days and three weeks, hybridoma cells are visible and culture supernatants may be screened for specific antibody secretion by a number of techniques including radioimmunoassay, enzyme-linked immunoassay, western blotting or immunostaining. Those culture wells containing hybridoma cells secreting an appropriate antibody are then expanded and 'subcloned' by limiting dilution or in soft agar to ensure that they are derived from a single progenitor cell (i.e. they are clonal) and to ensure stability of antibody production. Many hybridoma cells lose chromosomes and become unable to produce antibody soon after fusion. The subclones are, in turn, screened, and selected clones are expanded for antibody production in vitro (culture flasks or bioreactors) or in vivo as ascites in mice. Human hybridomas are produced in essentially the same fashion. However, some key differences remain, most importantly that, for ethical reasons, human subjects may only be immunized with a very

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Fig. 1 Improved immunization protocols

limited number of immunogens (mostly after vaccinations or naturally occurring infections). In-vitro immunization protocols (see below) have not been as efficacious in human systems as they have been in murine systems although the immortalization of h u m a n B cells prior to fusion with Epstein-Barr virus (EBV) has enabled production of human hybridomas specific for antigens administered in vitro 8. The procedure is inefficient, however, and relatively large numbers of undesired clones are produced. In addition, human peripheral blood lymphocytes, which have been the primary source of human B cells, are inferior to those derived from much scarcer spleen, tonsil, or lymph node tissue 9. Efficient generation of human hybridoma cells for general use awaits the further development of in-vitro immunization protocols.

Advances in immunization protocols Suppression of dominant responses In many instances the mammalian B cell repertoire is 'skewed' in favor of a relatively few immunodominant epitopes 1°, and antibodyforming cells with specificity for non-dominant epitopes may be rare after fusion. To maximize production of non-dominant monoclonal antibodies, a strategy has been devised 12 that relies upon the selective ability of the alkylating agent cyclophosphamide to dampen the immune response to a particular antigen, perhaps by killing antigen-stimulated blast cells (and other proliferating cells) or, alternatively, by interfering with the normal anti-idiotypic network. Thereafter, immunization with a similar antigen permits expression of a different set of clones. When mice were immunized with a homogenate of neonatal rat cerebellum followed by cyclophosphamide treatment and a second immunization with 11-day-old rat cerebellum, 85% of the mAbs resulting from fusion were specific for the second immunogen, i.e. 11-day-old rat cerebellum. In contrast, no selective antibodies were obtained following an identical immunization schedule omitting cyclophosphamide 1~. Similarly, this approach was successfully

• Suppression of dominant responses -- increases the chances of producing antibody-forming cells with specificity for rare epitopes or poor immunogens • In-vitro immunization -- can increase reproducibility of immunization and reduce time needed to produce antibody-forming cells, and permits generation of human antibody-producing cells • Antigen targeting -- can increase immunogenicity of antigens, easing the production of antibodyforming cells specific for poorly immunogenic or scarce antigens

Improvements in fusion • Electrofusion -- increases the frequency of hybrid cell formation 30--100 fold • Electrofusion with antigen bridging -- increases the frequency of formation of hybrid cells specific for a particular antigen • Laser fusion -- selected pairs of cells can be fused, reducing time needed to select or screen cells post fusion ~

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• Fusomas -- hybrid hybridoma cells can produce antibody molecules with specificity for two different antigens

Recent improvements in B-cell hybridoma production. The conventional techniques of hybridoma cell production depend on the mixing, of large numbers of spleen cells from immunized animals with non-secreting myeloma or hybridoma cells. Improvements address deficiencies in the immunization steps, fusion protocol and product range of the conventional technology.

used by us to produce mAbs to weakly immunogenic glycoaminoglycans and idiotypic antibodies. A similar but perhaps more direct approach is to use antibodies directed against unwanted antigens 12. These methods should both prove useful to investigators seeking mAbs to rare epitopes or poor immunogens and herald the development of new

methods of mAb generation based upon cogent manipulation of the immune system. In-vitro immunization In-vitro immunization offers a possible means of circumventing some of the difficulties encountered with conventional (in-vivo) immunization schemes 5'13, difficulties such

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as those of reproducibility, long delays between initial immunization and boost (three to four weeks), and, in humans, preclusion of immunization. Primary immunization of isolated lymphoid tissue was first performed in 1912 by Carrel and Ingebritsen TM, and systems for the stimulation of murine splenocyte preparations in vitro were well developed by the late 1960s ~5. Research based upon these and related systems has contributed greatly to our understand-, ing of lymphocytic interactions, growth factors and the propagation of lymphocytes in vitro, and has fostered methods to maximize the stimulation of B cells in vitro prior to fusion 16. In the most successful of these techniques, spleen cells from nonimmunized mice are incubated in complete growth medium 'conditioned' by the culture of murine thymus cells (thymocytes). The spleen cells are then immunized in vitro with small quantities of antigen. The factors secreted by the thymocytes facilitate the triggering of those B cells capable of binding and being activated by the immunizing antigens. Numerous large blast cells are visible four to five days post stimulation, at which time fusion is optimal 16. Additional improvements in in-vitro immunization will be particularly important in the generation of human hybridomas. Antigen targeting The immune response can be enhanced by the targeting of antigen to cells of the immune system. The immunogenicity of ferritin could be increased in mice by covalently coupling it to goat antibodies specific for murine IgG 17. Similarly, the immunogenicity of avidin and bovine serum albumin was enhanced by coupling them to anti-class II mAbs 18. These methods are likely to form the basis of protocols for the production of mAbs, especially where only small quantities of antigen are available or where the antigen (e.g. synthetic .peptide) is poorly immunogenic. The combination of antigen targeting and in-vitro immunization may also prove to be useful.

Electro fusion The pioneering work of Zimmermann and his colleagues 19 over the past 15 years has introduced a novel and powerful means of generating large numbers of antibodyforming hybrid cells. Their method is based upon the temporary disruption of living membranes when they are exposed to critical electrical field conditions. Zimmermann has termed this phenomenon 'reversible electrical breakdown' of the cell membrane. For most mammalian cell membranes, the 'breakdown' voltage is about 1 V a t 2 2 ° C a n d 2 V a t 4 °C for short duration pulses of 1-10 ~s and about half this amount for pulse durations in the 20-50 ~s range. Longer pulses tend to disrupt the membrane irreversibly and kill the cell. When two membranes are in apposition (in series), the voltages must be doubled so that both membranes are broken down. In the Zimmermann system, cells are first suspended in an iso-osmotic sugar solution, e.g. sorbitol with small amounts of Ca 2+ and Mg 2+, and introduced into a specially designed fusion chamber. The cells are then aligned using low-voltage alternating current to bring them into tight apposition before application of the fusion pulse. One or more 10-15 ~s direct current pulses of sufficient intensity to permit reversible breakdown are then administered. (The conditions must be determined empirically for each cell type.) The cells are allowed to recover for a few minutes, then grown and selected under standard conditions. The efficiency of fusion may be estimated by either fluorescence staining the nuclei of each of the fusion partners with, for example, Hoescht 33342 and hydroethidine before introducing them into the fusion chamber, or by using fusion partners with distinct surface markers, e.g. H-2D or H-2K, which may be recognized post fusion by dye-labeled mAbs. The percentage of double-stained cells may then be determined by fluorescence microscopy or by two-color FACS (fluorescence-activated cell sorting) analysis (size analysis is not reliable). Under optimal conditions, from 3% to 7% of viable splenic leukocytes

will be fused to SP2/0 immediately post fusion (G. A. N. and H. B. U., unpublished). For PEG-induced fusion, less than 0.5% (probably much less) of the splenic leukocytes are fused. FACS may also be used to recover hybrid cells post fusion as an alternative to chemical selection 2°. With these methods and standard immunization protocols, it was possible to obtain more than 2.5 stable dinitrophenol-specific, antibody-forming hybridoma cell clones per 104 splenic leukocytes fused, i.e. as many as 7000 hybridoma cell clones from a single immunized murine spleen (U. Zimmerman, unpublished), compared with 3-8.5 per 1 0 6 splenic leukocytes fused for unenhanced PEG fusion 5. The technology can be improved by incorporating liposomes or hydrophobic proteins in the fusion medium 21. Thus, electrofusion appears to be the method of choice for cheaply a n d efficiently generating large numbers of hybridomas. The major disadvantage of this system is that attaining high yields often requires both an initial time investment to create optimal conditions for the hybridoma or myeloma fusion partners and an initial investment in equipment. Antigen bridging Lo et a]. 22 introduced a potentially valuable supplement to the technique of electrofusion. They coupled antigen to avidin using 1,5-difluoro2,4-dinitrobenzene (DFDNB) and biotinylated the cell membranes of myeloma fusion partners using Nhydroxysuccinimidyl biotin 22. The spleen cells and myelomas were then incubated together prior to fusion to allow antigen-specific B cells to be 'bridged' to myeloma cells before applying the electrical pulse. The frequency of specific antibodysecreting hybrids (for angiotensin converting enzyme) was greatly increased and the resulting antibodies were enriched for high-affinity IgGs. Similar success with antigen bridging and electrofusion was reported by Wocjchowski and Sytkowski 23, with all resulting hybrids being specific for the immunizing antigen. A similar strategy 24'25 using PEG fusion attained frequencies of about 10 -5 (identical to the frequency

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obtained without targeting), but only about 12% of these were specific for the immunizing antigen (azobenzene arsonate), compared with 7% of nontargeted controls. The numbers of IgG-producing clones obtained were about the same, but once again increased affinity was demonstrated for the hybridomas produced by antigen bridging. Overall, it seems that this technique holds great promise for the efficient production of highaffinity mAbs, although it appears to be best suited to electrofusion or other methods (e.g. laser fusion) that obviate the nonspecific adhesiveness inherent in the PEG fusion procedure.

readily followed with this method, and the resulting hybridoma cells are easily identified and transferred to microwell plates using a micromanipulator. The laser fusion method is expensive and novel, but it may prove useful for certain specialized purposes requiring preselection of antibody-producing clones. Fusomas

One of the most exciting n e w developments in the production of mAbs is the fusion of two antibodysecreting hybridomas of known specificity, which results in a hybrid hybridoma or a 'fusoma' that secretes Laser fusion bispecific mAbs, i.e. a single imThe major disadvantage of existing munoglobulin comprised of two cell fusion techniques is not their distinct heavy and light chains and relative inefficiency, as it is usually having the antigen-binding capacity possible to produce large numbers of of both parents. The idea of using antibody-secreting clones especially bispecific antibodies to link two when electrofusion methods are distinct antigens predates hybridoma employed, but rather that they must technology. Chemically engineered be carried out in 'bulk', that is, a bivalent antibodies were first used minimum of 106 cells must undergo for staining tissue sections with the fusion process simultaneously TM. ferritin in 1968 by Hammerling et (/].27 Difficulties in preparing and Since only a few B cells per thousand are specific for a given immunogen, purifying these antibodies prevented a large number of hybridoma cell widespread adoption of the techclones specific for other antigens are nique, but the advent of hybridoma also found. Subsequent selection of technology has circumvented many the desired clones by screening and of these difficulties. subcloning is time-consuming and K6hler and Milstein observed that tedious. when two antibody-forming cells are If it were possible to select anti- fused, the derived hybrid continues body-producing B-cell clones prior to show expression of the immunoto fusion and then to fuse only globulin chains of its parents, i.e. those clones desired with a high allelic exclusion does not apply to probability of success, post-fusion these cells as it does for naturally selection w o u l d be obviated. Methods occurring antibody-forming cells 1. to achieve this goal are currently Subsequent studies 28 have shown under development. Among the most that the heavy and light chains are promising of these is laser-induced independently assembled by random cell fusion 26 in which B-cell- association into immunoglobulins in myeloma-cell conjugates prepared the cytoplasm. using the avidin-biotin antigenDiffering rates of chain synthesis bridge method 22 are, fused using an and the preference of certain chains excimer laser emitting 17 ns pulses at to associate often dictate that the final 308 nm with a repetition rate of 1- ratios of the various antibodies will 100 Hz used to 'pump' a dye laser not be constant and will affect the tunable from 320-970 nm. The dye yield of bispecific antibodies 29. For laser beam is directed into the example, ~t and 7 heavy chains do fluorescence illumination path of an not usually associate, hence the inverted microscope, with a final expected yield of such a bispecific beam diameter of 0.3-0.5 ~tm. The mAb would be negligible. Urnovitz target cell pairs are identified and e t al. have recently described irradiated with 300-600 (1 ~tJ) pulses how naturally occurring, polymeric at 340 nm. The fusion process can be immunoglobulins (IgA and IgM)

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can form hybrid polymers, that is, IgA to IgM monomers linked, most probably, via their penultimate cysteine residues 3°. Unlike conventional mAbs, the mAbs resulting from this process are heterogeneous (a mixture of homoand heteropolymers) and one or more purification steps (e.g. DEAE or hydroxyapatite chromatography 31,32) are required to achieve homogeneity. Post-fusion selection of hybrid hybridomas may be performed by drug selection. For instance, one partner may be rendered HAT sensitive and ouabain resistant by selection in 8azaguanine and ouabain-containing medium, and the other HAT resistant and ouabain sensitive (as is the case for most hybridomas) 32. Alternatively, fused hybridomas can be successfully selected by FACS 2°. The utility of bispecific mAbs in immunoassays and immunostaining is n o w being realized. Reagents capable of simultaneously binding peroxidase and somatostatin 31 and afetoprotein 2° are among those n o w in use. The principal advantage of these reagents is that chemical coupling with its attendant loss of activity and increase in size (which may inhibit tissue penetration) may be avoided and the sensitivity of the assay may be increased. Perhaps the most exciting application of fusoma technology has been the generation of bivalent mAbs with specificity for the T-cell receptor complex and tumor antigens 3. Like their chemically linked counterparts, these reagents are able to activate and direct cytotoxic T lymphocytes to their targets with consequent efficient cytolysis. It is hoped that this approach will prove to be a useful means of killing tumor cells i n vitro and hence a valuable adjunct therapy in cancer treatment. More research

Although the technique (some would say 'art') of hybridoma technology has become a discipline in its own right, it has at the same time become increasingly integrated into nearly every field of biology and medicine. Continued research that focuses on understanding the events that lead to the viable fusion of cell partners (e.g. mixing of adjacent cell

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m e m b r a n e s , h y b r i d nuclei formation, cell recovery) will be the area that converts this h y b r i d o m a cell techn o l o g y from art to science. The resulting innovative techniques s h o u l d c o n t r i b u t e to the full realization of the e n o r m o u s potential of m o n o c l o n a l antibodies.

Acknowledgement We t h a n k N a d i n e Williams Martin for her t e c h n i c a l editing expertise.

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References 1 K6hler, G. and Milstein, C. (1975) Nature 256,495-497 2 Miller, R. A., Maloney, D. G., Warnke, R. and Levy, R. (1982) N. Engl. J. Med. 306, 517-522 3 DePinho, R. A., Feldman, L. B. and Scharff, M.D. (1986) Ann. Intern. Med, 104, 225-233 4 Fazekas de St Groth, S. and Scheidegger, D. (1980) ]. Immunol. Methods 35, 1-21 5 Galfre, G., Howe, S., Milstein, C., Butcher, G. and Howard, J. (1977) Nature 266, 550-552 6 Leo, O., Foo, M., Sachs, D. H., Samelson, L.E. and Bluestone, J. A. (1987) Proc. Natl Acad. Sci. USA 84, 1374-1378 7 Shulman, M., Wilde, C. D. and K6hler, G. (1978) Nature 276,269-270 8 Yamaura, N., Makino, M., Walsh, L.J., Bruce, A.W. and Choe, B.K. (1985) J. Immuno]. Methods 84, 105-116 9 James, K. and Bell, G. T. (1987) J. Immunol. Methods 100, 5-40 10 Neil, G. A. and Klinman, N. R. (1987) Int. Rev. Immuno]. 2, 307-320 11 Chiu, A. Y., Matthew, W. D. and Patterson, P.H. (1986) f. Cell. Biol. 103, 1383-1398 12 Thalhamer, J. and Freund, J. (1985) J. Immunol. Methods 80, 7-13 13 Matthew, W. D. and Sandrock, A. W. (1987) J. Immunol. Methods 100, 73-82 14 Carrel, A. and Ingebritsen, R. (1912) J. Exp. Med. 15, 287-291 15 Mishell, R. I. and Dutton, R. W. (1967) J. Exp. Med. 126, 423-442 16 Luben, R. A. and Moller, M. A. (1980) Mol. Immunol. 17, 635-639 17 Kawamura, H. and Berzofsky, J.A. (1986) J. Immunol. 136, 58-65 18 Carayanniotis, G. and Barber, B.H. (1987) Nature 327, 59-61 19 Zimmermann, U. and Urnovitz, H. B. (1988} MethodsEnzymol. 15,194-221 20 Karawajew, L., Micheel, B., Behrsing, O. and Gaestel, M. (1987)J. Immunol. Methods 96, 265-270 21 Ohnishi, K., Chiba, J., Goto, Y. and

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Tokunaga, T. (1987) J. Immunol. Methods 100, 181-189 Lo, M. M. S., Tsong, T. Y., Conrad, M. K., Strittmayer, S. M., Hester, L. D. and Snyder, S. H. (1984) Nature 310, 792-794 Wocjchowski, D. M. and Sytkowski, A.J. (1986) J. Immunol, Methods 90, 173-177 Bankert, R. B., DesSoye, D. and Powers, L. (1980) Transplant. Proc. 12, 443-446 Reason, D., Carminati, J., Kimura, J. and Henry, C. (1987) J. Immuno]. Methods 99, 253-257 Wiegand, R., Weber, G., Zimmermann, K., Monajembashi, S., Wolfrum, J. and

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Greulich, K. (1987) ]. Cell Sci. 88, 145-149 Hammerling, U., Aoki, T., de Harven, E., Boyse, E. A. and Old, C. J. (1968) J. Exp. Med. 128, 1461-1469 Cotton, R. G. H. and Milstein, C. (1979) Nature 244, 42-43 Suresh, M. R., Cuello, A. C. and Milstein, C. (1986) Methods Enzymo]. 121,210-228 Urnovitz, H., Chang, Y., Scott, M., Fleischman, J. and Lynch, R. (1988) J. Immunol. 140,558-563 Milstein, C. and Cuello, A. (1983) Nature 305, 537-540 Staerz, U. and Bevan, M. (1986) Proc. Nat] Acad. Sci. USA 83, 1453-1457

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Nucleic acid hybridization in plant virus diagnosis and ch a ra cte rizati o n Roger Hull and Ali AI-Hakim The identification of viruses is important in the prediction of plant diseases in annual crops, the prevention of infection in planting stock, in monitoring disease control methods and in diagnosing disease in plants held in quarantine. Nucleic acid hybridization methods are well suited to such purposes. Their applicability will be extended by developments in membrane materials, probes and non-radioactive reporter groups. R e c o m b i n a n t DNA t e c h n o l o g y is proving to be a v e r y p o w e r f u l tool in plant virology. Not o n l y are g e n o m e organizations and gene functions being d e t e r m i n e d but it is giving n e w a p p r o a c h e s to virus diagnosis and the d e t e r m i n a t i o n of relationships bet w e e n viruses. These n e w a p p r o a c h e s d e p e n d u p o n the h y b r i d i z a t i o n of a probe n u c l e i c acid to the target viral nucleic acid. The probe comprises sequences that are c o m p l e m e n t a r y to the target and to w h i c h r e p o r t e r groups are attached. Since the c o m p o s i t i o n of the probe nucleic acid and the

parameters of h y b r i d i z a t i o n can be varied, this a p p r o a c h is flexible. T h e major aim of the use of this techn o l o g y in diagnosis is to p r o v i d e a simple, sensitive, reliable system.

Uses of plant virus diagnostics T h e r e are several different situations in w h i c h viral diagnosis is n e e d e d . For a n n u a l crops, the m a i n use of diagnosis is in disease prediction. Viruses spread b y different agents can cause similar s y m p t o m s ; c o n v e r s e l y s y m p t o m s due to a given agent can vary w i t h crop variety. Thus, the virus m u s t be identified a c c u r a t e l y and r a p i d l y so that its m o d e of t r a n s m i s s i o n can be u n d e r stood a n d control m e a s u r e s effected. This is b e c o m i n g even m o r e important as farmers r e d u c e inputs for

Roger Hull and Ali Al-Hakim are at the John Innes Institute, AFRC Institute of Plant Science Research, Colney Lane, Norwich, UK. © 1988,

Elsevier Publications, Cambridge

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