Immunology Today, voL 7, No. 1 I, 1986
-r vl ,liV$
The productionof more useful monoclonalantibodies I. Modificationsof the basictechnology Hybridoma technology has greatly expanded the usefulness and application of immunoassaysand created the potential for in-vivo immunodiagnosis and therapy. While the basic technique has not changedappreciably,a number of modifications have been introduced which make it possible to obtain more useful monoclonal antibodies and to generate monoclonals against weakly immunogenic substances. In this first part of a two-part review, Matthew Scharff and his colleagues review the most useful of these modifications in the production of animal monodonal antibodies, paying particular attention to the problem of raising antibodies against small amounts of weak immunogens. Ten years ago Kohler and Milstein 1 described the hybridoma technology for generating monoclonal antibodies (mAb). From the outset there was no question that mAb would significantly improve the quality and reliability of immunoassays. The ability to generate large amounts of a chemically homogeneous antibody which could be replenished whenever it was needed also led to the conviction that mAb would be useful clinical reagents. However, a number of problems have delayed the full realization of their potential for diagnosis and treatment of human disease in vivo. Examination of the serum of an immunized animal often reveals that some of the antibodies the animal is producing are more specific and have higher affinities than the rest. Improvements in the existing technology which would more completely sample the whole repertoire of antibody forming cells could provide mAb of the desired affinity, specificity, or function. Once the best natural product is recovered, its usefulness could be improved either by using somatic cell genetics to generate mutant antibodies or by generating novel reagents through the structural manipulation of the antibody genes. In essence, the current generation of mAb is like the early antibiotics which made it possible to generate still more useful second and third generation reagents. In the first part of this two-part review, we will identify some of the persisting problems in generating mouse and rat mAb and indicate the existing solutions to these problems. In Immunology Today next month we will review the use of somatic cell genetics and the recombinant DNA technology to generate even better reagents. Relatively few studies have systematically explored and modified each of the parameters of hybridoma technology 2, probably because the immune response is unpredictable even in inbred animals immunized with the same antigen. This makes it almost impossible to compare results with different antigen-antibody systems or even from one fusion with the next. Nevertheless, we have tried to formulate a series of approaches aimed at obtaining the required mouse and rat mAb as efficiently as possible.
344
AlbertEinsteinCollegeof Medicine,Bronx,New York 1046I, USA.
Deborah French, Ellyn Fischberg, Susan Buhl and Matthew D. Scharff Immunization strategies Before producing a set of mAb, it is important to define their potential uses. For example, if they are to be used on glutaraldehyde fixed material, then the animals should be immunized and the fusion screened with fixed antigen. It is also important to recognize that a large number of hybrids will have to be screened over a short period of time therefore sufficient antigen and an appropriate screening assay must be available. Many different immunization schemes have been proposed 3, and different protocols may work better for different immunogens. In general we try to use the simplest immunization scheme available. For soluble antigens, we usually start out with a single injection of 50-100 p,g of antigen in complete Freund's adjuvant intraperitoneally, bleed the animals at 4 weeks to determine the quality and quantity of antibody generated, rest the animals until the titer has dropped and then boost with 100 I~g of antigen in saline intravenously and fuse 3 days later. If Freund's adjuvant is used, it may be more important to inject highly concentrated antigen than to introduce large absolute amounts 4. Also the injected material must be a true water-in-oil emulsion 5. If the antigen of interest is highly immunogenic, almost any immunization scheme will stimulate the generation of many antibody forming cells and ultimately many useful hybridomas. However, since the frequency of hybridomas producing the desired antibodies is roughly related to the number of antibody forming cells present at the time of fusion, antigens that elicit a weak response because they are weakly immunogenic or available in very small amounts present the greatest problems. A common and often successful way to increase the immunogenicity of such antigens is to use glutaraldehyde to conjugate the antigen with a highly immunogenic carrier such as keyhole limpet hemocyanin 6. This increases immunogenicity probably by a combination of chemical modification and recruitment of carrier-specific helper T cells. It also carries the risk of immunizing against denatured sites. Another, perhaps more benign, way of increasing immunogenicity is to adsorb the antigen to nitrocellulose. There is a preliminary report that effective immunization can be carried out with as little as 10 ng of protein if it is adsorbed to nitrocellulose and the nitrocellulose is inserted into the peritoneal cavity of the animal 7. While effective immunization with such small amounts of antigen has yet to be confirmed, larger amounts of antigen have been adsorbed to nitrocellulose which can be reduced to small particles with dimethylsulphoxide (DMSO) and injected 8. Small amounts of antigen can also be injected directly into the spleen either as free antigen 9 or adsorbed to nitrocellulose which has been homogenized into small particles (S. Lithicum, unpublished). ~) 1986, ElsevierSciencePublishers B.V, Amsterdam 0167-4919/86/$02.00
Immunology Today, vol. 7, No. 1I, 1986
We always try to immunize 5-10 animals and then, at the expected peak of the response, bleed and test their sera for the presence of the required antibody. For example, if the goal is to make anti-idiotypic mAb specific for the antigen binding site of a particular immunoglobulin, then it is essential to identify those animals making the highest titer of antibody whose binding is inhibited by antigen. In some cases, it is necessary to adsorb irrelevant antibodies from the serum and then test the residual titer for the desired antibody. Thus if antibody that reacts with a particular tumor cell is needed, it may be useful to adsorb the serum antibody with relevant normal cells and then assay for the titer of residual tumor reactive antibodies. While there are certain dangers in depending upon an initial titre to predict a subsequent response, we believe that the chances of success are increased if the animals making the highest titer of the desired antibody are rested and then used for the fusion. When should cell fusion be undertaken? Some workers boost the animals for the fusion while there are still high titers of circulating antibody. Protocols such as the one described by St~hli 1° which involves the repeated injection of 300-400 I~g of antigen may decrease the titers of circulating antibody and allow antigento be presented appropriately. However, our experience suggests that, at least with protein antigens, the maximum number of antibody producing hybridomas will be obtained if the animals are rested until there is little circulating antibody, and then boosted for the fusion.
Fusion A number of fusion protocols use polyethyleneglycol to promote fusion. We have found the protocol described by Fazekas de St Groth 1~ to be reproducible and effective. Most workers fuse 3-4 days after the boost and there is some evidence that the frequency of blast cells determines the number of hybrids 1°'~2. Since very few hybrids are generated five days after a boost, four days may also be too late in some cases and we therefore prefer to fuse at three days. In fact, Kubagawa et aL ~3 suggested that lymph node fusions be carried out one day after the boost. We expect a yield of at least 2000 and usually 5000 hybrids/108 spleen cells. Some investigators use thymocytes or macrophages as feeders to increase the cloning efficiency of the hybridomas. However, we have found that feeders are unnecessary if the serum being used supports a cloning efficiency of the parental myeloma cells of better than 80% when assayed by limiting dilution. Such 'cloning' sera are then compared in active fusions for the number of hybridomas that they will support and the best lot is saved for fusions. Occasional batches of foetal calf sera promote the growth of small attached round cells or fibroblasts and macrophages. If such cells rapidly cover the well, they may retard or even inhibit the outgrowth of hybridomas, so such sera are not used for fusions. It is also worth noting that media filtered through 'Triton' treated filters ~4 (for example 'Millipore' filters are triton treated and 'Millex' are not) and media exposed for moderate periods of time to ordinary fluorescent lights inhibit the growth of clones ~5. A number of serum free media are now available which facilitate the purification of mAb from tissue culture media.
reviewsEnrichment of antibody forming cells Many hundreds of hybridomas making the desired antibody should be obtained from a single fusion if the spleen cells are from an animal immunized with a highly immunogenic antigen. However, there are still problems in obtaining the desired mAb from animals immunized with weakly immunogenic or small amounts of antigen. For these situations, it is often necessary to specifically enrich the spleen cell culture for the B cells making the desired antibody. Spleen cells harvested for fusion three days after the boost can be reinjected into irradiated recipients along with antigen. This results in the selective amplification of specific B cells and as much as a 50 fold increase in hybridomas making the desired antibody 16. In fact, with weak immunogens one can divide the spleen cells obtained three days after the boost and inject 4 x 106 cells/mouse into four irradiated mice and use the remaining spleen cells for a standard fusion. Using the protocol described by Siraganian et al. ~6, cells from the repopulated spleens are also fused. With weak immunogens, we frequently obtain only a few positive hybrids from the standard fusions and many from the repopulated fusion. It is also possible to enrich for cells making the desired antibody and to make antibodies against highly conserved antigens by immunizing in vitro. This may be done either with spleen cells from immunized animals or as a primary immunization. Here again a number of protocols have been published ~7'18. We have found the one described by Boss19 simple to use because it employs a commercially available adjuvant peptide rather than various T-cell factors. The approach described by Van Ness et aL2° uses silica to present antigens and does not require foetal calf serum. This may be a useful way to deal with inherently insoluble antigens that otherwise require detergents. It is not clear why some antigens do not elicit an antibody response in vivo but do in vitro. However, this does occur and antibody producing hybridomas can be generated using small amounts of antigen and primary immunization in vitro. Selective fusion of B cells making the desired antibody results in only a few hybrids, almost all of which are producing the required antibody. These techniques use 'antigen focused' fusion in which the antigen is covalently attached to the surface of the myeloma fusion partner. These antigen bearing myeloma cells are then mixed with immune spleen cells which express antibody on their surface. Aggregates are formed between the desired antibody forming cells and the myeloma parent and fusion is promoted by polyethyleneglyco121 or electrofusion 22. This technique is reported to promote the fusion of cells making the desired antibody 23'24, greatly reducing the number of hybrids that have to be screened, but it has not been widely used suggesting that there are difficulties in its successful application. The recent modifications of the technique reported by Lo et a/. 25 use avidin and biotin to increase the affinity of binding of the two cell types. The hybrids generated make high affinity antibodies suggesting that antigen focused fusion may require B cells with high affinity receptors, thus making it possible to selectively fuse the B cells making the most desirable antibodies. Isolation of hybridomas making antibody against the non-immunodominant epitopes in a mixture of antigens or on a complex antigen can be facilitated by selectively suppressing the response to the immunodominant anti-
345
Immunology Today, voL 7, No. 11, 1986
gen by simultaneously priming the animal with antigen and the cytotoxic agent cyclophosphamide. Subsequent immunization with the same mixture should then favour minor antigenic determinants 26. Passive immunization with antibodies against one antigen in a mixture may also suppress the subsequent response to that antigen 27, although this will probably depend upon the ratio of antigen and antibody. If used together, the various modifications of the hybridoma technique make it possible to obtain mAb even from weak immunogens that are available in small quantities. Routine use of antigen focused fusion greatly reduces the amount of screening required. Although numerous techniques have been used by cellular immunologists to enrich for B cells making a particular antibody, none of these has been used routinely to improve the hybridoma technology. On the other hand, if the efficiency of fusion was increased, for example using electrofusion 2s'28, then more screening would be required in order to sample the repertoire more completely and to identify rare antibodies. It may also be possible to increase fusion frequency by attempting to synchronize the cell cycles of either or both the spleen and myeloma populations 2°,29. Screening
Finally, it is worth re-emphasizing the need to screen fusions for antibodies with the desired properties quickly and then to clone the relevant hybrids as rapidly as possible. While it is always tempting to try to recover and clone all of the hybridomas producing specific antibody, cloning and freezing is the most time consuming aspect of the process. It is therefore useful to identify the most useful hybridomas as quickly as possible. For example, if high titers and high affinity antibodies are needed, the media from the initial hybridoma wells can be diluted 10 or even 100 fold and then screened. Many slower growing or low producing hybridomas will be missed but those can be identified by rescreening the original wells. With certain antigens, only high affinity antibodies will bind to ELISA plates containing a low density of antigen 3°. If antibodies of a certain class or subclass are needed, they can be screened directly by using antisera specific for that isotype. The instability of hybrids can be a problem but even very unstable hybridomas can be identified by cloning early and recloning repeatedly31. Instability can be reduced by using the system described by Taggart and Samloff32 in which the immunoglobulin heavy chain gene and the gene encoding resistance to a particular drug are on the same chromosome.
Conclusions
346
The quality and quantity of mouse and rat monoclonal antibodies can be improved by increasing immunogenicity, selecting the best animals, enriching for B cells making the desired antibody, increasing the efficiency of fusions, and screening judiciously. Additional improvement in each of these steps would be useful and may ultimately make it possible to recover the best and most useful antibodies which the individual is capable of producing. Such innovations may also make it possible to generate routinely human monoclonal antibodies which are still difficult to obtain and which would be very useful for diagnosis and therapy in vivo. In addition, there
would be many more uses of antigen binding sites if the structure of the antibody could be appropriately modified and this will be discussed in the second part of this article. Someof the work describedabovewas supportedby grantsfromthe NIH (AI105231)and NSF(DCB8316150).The HybridomaFacilityat Einsteinis supportedbya CancerCenterGrant(2P30CA13330).DeborahFrenchisaFellow of the NewYorkHeartAssociationandallof theauthorsarepartof theIrvington HouseInstitutefor MedicalResearch.Wewouldliketo thankDrJosephDaviefor hishelpfulsuggestions. References
1 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 2 Westerwoudt, R.J.(1985)J. ImmunoL Meth. 77, 181 196 3 Damjanov, I. and Knowles, B.B.(1983)Lab. Invest. 48, 510-525 4 Farr,R.S. and Dixon F.J.(1960)./.Immuno/. 85, 250-257 5 Weir, D.M. (ed.) (1973) Applications oflmrnunological Methods (2nd edn), Appendix 2, Blackwell Scientific
Publications, Oxford 6 Sakato, N. and Eisen,H.N. (1975)J. Exp. Med. 141, 1411-1425 7 Sternick,J.L. and Sturmer, A.M. (1984) Hybridoma 3,74 (abs.) 8 Knudsen, K.A. (1985)Anal Biochem. 147,285-288. 9 Spitz, M, Spitz, L., Thorpe, R. eta/. (1984)J. Immuno/. Meth. 70, 39-43 10 Stahli, C., Staehelin,T., Miggiano, V. eta/. (1980) J. ImmunoL Meth. 32,297-304 11 Fazekasde St Groth, S. and Scheidegger, D. (1980) J. Immuno/. Meth. 35, 1-21 12 Andersson,J. and Melchers, F. (1978) Curr. Top. Microbiol. Immuno/. 81, 130-139 13 Kubagawa, H., Mayumi, M, Kearney,J.F. eta/. (1982) J. Exp. Med. 156, 1010-1024 14 Cahn, R.D. (1967)Science 155, 195-196 15 Wang, RJ. (1976)/n Vitro 12, 19 22 16 Siranganian,R.P.,Fox, P.C.and Berenstein, E.H. (1983) Meth. Enzymo/. 92, 17-26 17 Reading,C.L. (1982)J. Immuno/. Meth. 53, 261-291 18 Rathjen, P.A. and Underwood, P.A. (1985)J. ImmunoL Meth. 78, 227-237 19 Boss,B.D. (1983) Brain Res, 193-196 20 Van Ness,J., Laemmli, U.K. and Pettijohn, D.E. (1984) Proc. Natl Acad. Sci. USA 81, 7897-7901 21 Gefter, M.L. MargulJes,D.H. and Scharff, M.D. (1977) Somatic Cell Genet. 3, 231-236 22 Zimmerman, U. (1982) Biochem. Biophys. Acta 694, 227-277 23 Bankert, R.B., Dessoye,D. and Powers, L. (1980) Transplant. Proc, 12,443--446 24 Kranz, D.M., Billing, P.A., Herron, J.N. eta/. (1980) Immunol. Commun. 9, 639-651 25 Lo, M.M.S., Tsong, T.Y., Conrad, M.K. eta/. (1984) Nature (London) 31 O, 792-794 26 Matthew, W.D. and Patterson, P.H. (1983) ColdSpring Harbor Syrup. Quant. BioL XLVIII,625-631 27 Thalhamer,J. and Freund, J. (1985)J. Immuno/. Meth. 80, 7-13 28 Karsten,U., Papsdorf, G., Roloff, G. eta/. (1985) Eur. J. Cancer C/in. Onto/. 21,733-740 29 Miyahara, M., Nakamura, H. and Hamagochi, Y. (1984) Biochem. Biophys. Res. Comm. 124, 903-908 30 Herzenberg, L.A., Black, S.J.,Takuhisa,T. eta/. (1980)J. Exp. Med. 151, 1071-1087 31 Zack, D.J. and Scharff, M.D. (1984)in Single Cell Monitoring Systems (Ansari H.A. and DeSerresF.J.eds) pp. 233-263, Plenum Publishing Corporation, London 32 Taggart, R.T.and Samloff, I.M. (1983) Science 219, 1228-1230
-r vl ,liV$
The productionof more useful monoclonalantibodies I. Modificationsof the basictechnology Hybridoma technology has greatly expanded the usefulness and application of immunoassaysand created the potential for in-vivo immunodiagnosis and therapy. While the basic technique has not changedappreciably,a number of modifications have been introduced which make it possible to obtain more useful monoclonal antibodies and to generate monoclonals against weakly immunogenic substances. In this first part of a two-part review, Matthew Scharff and his colleagues review the most useful of these modifications in the production of animal monodonal antibodies, paying particular attention to the problem of raising antibodies against small amounts of weak immunogens. Ten years ago Kohler and Milstein 1 described the hybridoma technology for generating monoclonal antibodies (mAb). From the outset there was no question that mAb would significantly improve the quality and reliability of immunoassays. The ability to generate large amounts of a chemically homogeneous antibody which could be replenished whenever it was needed also led to the conviction that mAb would be useful clinical reagents. However, a number of problems have delayed the full realization of their potential for diagnosis and treatment of human disease in vivo. Examination of the serum of an immunized animal often reveals that some of the antibodies the animal is producing are more specific and have higher affinities than the rest. Improvements in the existing technology which would more completely sample the whole repertoire of antibody forming cells could provide mAb of the desired affinity, specificity, or function. Once the best natural product is recovered, its usefulness could be improved either by using somatic cell genetics to generate mutant antibodies or by generating novel reagents through the structural manipulation of the antibody genes. In essence, the current generation of mAb is like the early antibiotics which made it possible to generate still more useful second and third generation reagents. In the first part of this two-part review, we will identify some of the persisting problems in generating mouse and rat mAb and indicate the existing solutions to these problems. In Immunology Today next month we will review the use of somatic cell genetics and the recombinant DNA technology to generate even better reagents. Relatively few studies have systematically explored and modified each of the parameters of hybridoma technology 2, probably because the immune response is unpredictable even in inbred animals immunized with the same antigen. This makes it almost impossible to compare results with different antigen-antibody systems or even from one fusion with the next. Nevertheless, we have tried to formulate a series of approaches aimed at obtaining the required mouse and rat mAb as efficiently as possible.
344
AlbertEinsteinCollegeof Medicine,Bronx,New York 1046I, USA.
Deborah French, Ellyn Fischberg, Susan Buhl and Matthew D. Scharff Immunization strategies Before producing a set of mAb, it is important to define their potential uses. For example, if they are to be used on glutaraldehyde fixed material, then the animals should be immunized and the fusion screened with fixed antigen. It is also important to recognize that a large number of hybrids will have to be screened over a short period of time therefore sufficient antigen and an appropriate screening assay must be available. Many different immunization schemes have been proposed 3, and different protocols may work better for different immunogens. In general we try to use the simplest immunization scheme available. For soluble antigens, we usually start out with a single injection of 50-100 p,g of antigen in complete Freund's adjuvant intraperitoneally, bleed the animals at 4 weeks to determine the quality and quantity of antibody generated, rest the animals until the titer has dropped and then boost with 100 I~g of antigen in saline intravenously and fuse 3 days later. If Freund's adjuvant is used, it may be more important to inject highly concentrated antigen than to introduce large absolute amounts 4. Also the injected material must be a true water-in-oil emulsion 5. If the antigen of interest is highly immunogenic, almost any immunization scheme will stimulate the generation of many antibody forming cells and ultimately many useful hybridomas. However, since the frequency of hybridomas producing the desired antibodies is roughly related to the number of antibody forming cells present at the time of fusion, antigens that elicit a weak response because they are weakly immunogenic or available in very small amounts present the greatest problems. A common and often successful way to increase the immunogenicity of such antigens is to use glutaraldehyde to conjugate the antigen with a highly immunogenic carrier such as keyhole limpet hemocyanin 6. This increases immunogenicity probably by a combination of chemical modification and recruitment of carrier-specific helper T cells. It also carries the risk of immunizing against denatured sites. Another, perhaps more benign, way of increasing immunogenicity is to adsorb the antigen to nitrocellulose. There is a preliminary report that effective immunization can be carried out with as little as 10 ng of protein if it is adsorbed to nitrocellulose and the nitrocellulose is inserted into the peritoneal cavity of the animal 7. While effective immunization with such small amounts of antigen has yet to be confirmed, larger amounts of antigen have been adsorbed to nitrocellulose which can be reduced to small particles with dimethylsulphoxide (DMSO) and injected 8. Small amounts of antigen can also be injected directly into the spleen either as free antigen 9 or adsorbed to nitrocellulose which has been homogenized into small particles (S. Lithicum, unpublished). ~) 1986, ElsevierSciencePublishers B.V, Amsterdam 0167-4919/86/$02.00
Immunology Today, vol. 7, No. 1I, 1986
We always try to immunize 5-10 animals and then, at the expected peak of the response, bleed and test their sera for the presence of the required antibody. For example, if the goal is to make anti-idiotypic mAb specific for the antigen binding site of a particular immunoglobulin, then it is essential to identify those animals making the highest titer of antibody whose binding is inhibited by antigen. In some cases, it is necessary to adsorb irrelevant antibodies from the serum and then test the residual titer for the desired antibody. Thus if antibody that reacts with a particular tumor cell is needed, it may be useful to adsorb the serum antibody with relevant normal cells and then assay for the titer of residual tumor reactive antibodies. While there are certain dangers in depending upon an initial titre to predict a subsequent response, we believe that the chances of success are increased if the animals making the highest titer of the desired antibody are rested and then used for the fusion. When should cell fusion be undertaken? Some workers boost the animals for the fusion while there are still high titers of circulating antibody. Protocols such as the one described by St~hli 1° which involves the repeated injection of 300-400 I~g of antigen may decrease the titers of circulating antibody and allow antigento be presented appropriately. However, our experience suggests that, at least with protein antigens, the maximum number of antibody producing hybridomas will be obtained if the animals are rested until there is little circulating antibody, and then boosted for the fusion.
Fusion A number of fusion protocols use polyethyleneglycol to promote fusion. We have found the protocol described by Fazekas de St Groth 1~ to be reproducible and effective. Most workers fuse 3-4 days after the boost and there is some evidence that the frequency of blast cells determines the number of hybrids 1°'~2. Since very few hybrids are generated five days after a boost, four days may also be too late in some cases and we therefore prefer to fuse at three days. In fact, Kubagawa et aL ~3 suggested that lymph node fusions be carried out one day after the boost. We expect a yield of at least 2000 and usually 5000 hybrids/108 spleen cells. Some investigators use thymocytes or macrophages as feeders to increase the cloning efficiency of the hybridomas. However, we have found that feeders are unnecessary if the serum being used supports a cloning efficiency of the parental myeloma cells of better than 80% when assayed by limiting dilution. Such 'cloning' sera are then compared in active fusions for the number of hybridomas that they will support and the best lot is saved for fusions. Occasional batches of foetal calf sera promote the growth of small attached round cells or fibroblasts and macrophages. If such cells rapidly cover the well, they may retard or even inhibit the outgrowth of hybridomas, so such sera are not used for fusions. It is also worth noting that media filtered through 'Triton' treated filters ~4 (for example 'Millipore' filters are triton treated and 'Millex' are not) and media exposed for moderate periods of time to ordinary fluorescent lights inhibit the growth of clones ~5. A number of serum free media are now available which facilitate the purification of mAb from tissue culture media.
reviewsEnrichment of antibody forming cells Many hundreds of hybridomas making the desired antibody should be obtained from a single fusion if the spleen cells are from an animal immunized with a highly immunogenic antigen. However, there are still problems in obtaining the desired mAb from animals immunized with weakly immunogenic or small amounts of antigen. For these situations, it is often necessary to specifically enrich the spleen cell culture for the B cells making the desired antibody. Spleen cells harvested for fusion three days after the boost can be reinjected into irradiated recipients along with antigen. This results in the selective amplification of specific B cells and as much as a 50 fold increase in hybridomas making the desired antibody 16. In fact, with weak immunogens one can divide the spleen cells obtained three days after the boost and inject 4 x 106 cells/mouse into four irradiated mice and use the remaining spleen cells for a standard fusion. Using the protocol described by Siraganian et al. ~6, cells from the repopulated spleens are also fused. With weak immunogens, we frequently obtain only a few positive hybrids from the standard fusions and many from the repopulated fusion. It is also possible to enrich for cells making the desired antibody and to make antibodies against highly conserved antigens by immunizing in vitro. This may be done either with spleen cells from immunized animals or as a primary immunization. Here again a number of protocols have been published ~7'18. We have found the one described by Boss19 simple to use because it employs a commercially available adjuvant peptide rather than various T-cell factors. The approach described by Van Ness et aL2° uses silica to present antigens and does not require foetal calf serum. This may be a useful way to deal with inherently insoluble antigens that otherwise require detergents. It is not clear why some antigens do not elicit an antibody response in vivo but do in vitro. However, this does occur and antibody producing hybridomas can be generated using small amounts of antigen and primary immunization in vitro. Selective fusion of B cells making the desired antibody results in only a few hybrids, almost all of which are producing the required antibody. These techniques use 'antigen focused' fusion in which the antigen is covalently attached to the surface of the myeloma fusion partner. These antigen bearing myeloma cells are then mixed with immune spleen cells which express antibody on their surface. Aggregates are formed between the desired antibody forming cells and the myeloma parent and fusion is promoted by polyethyleneglyco121 or electrofusion 22. This technique is reported to promote the fusion of cells making the desired antibody 23'24, greatly reducing the number of hybrids that have to be screened, but it has not been widely used suggesting that there are difficulties in its successful application. The recent modifications of the technique reported by Lo et a/. 25 use avidin and biotin to increase the affinity of binding of the two cell types. The hybrids generated make high affinity antibodies suggesting that antigen focused fusion may require B cells with high affinity receptors, thus making it possible to selectively fuse the B cells making the most desirable antibodies. Isolation of hybridomas making antibody against the non-immunodominant epitopes in a mixture of antigens or on a complex antigen can be facilitated by selectively suppressing the response to the immunodominant anti-
345
Immunology Today, voL 7, No. 11, 1986
gen by simultaneously priming the animal with antigen and the cytotoxic agent cyclophosphamide. Subsequent immunization with the same mixture should then favour minor antigenic determinants 26. Passive immunization with antibodies against one antigen in a mixture may also suppress the subsequent response to that antigen 27, although this will probably depend upon the ratio of antigen and antibody. If used together, the various modifications of the hybridoma technique make it possible to obtain mAb even from weak immunogens that are available in small quantities. Routine use of antigen focused fusion greatly reduces the amount of screening required. Although numerous techniques have been used by cellular immunologists to enrich for B cells making a particular antibody, none of these has been used routinely to improve the hybridoma technology. On the other hand, if the efficiency of fusion was increased, for example using electrofusion 2s'28, then more screening would be required in order to sample the repertoire more completely and to identify rare antibodies. It may also be possible to increase fusion frequency by attempting to synchronize the cell cycles of either or both the spleen and myeloma populations 2°,29. Screening
Finally, it is worth re-emphasizing the need to screen fusions for antibodies with the desired properties quickly and then to clone the relevant hybrids as rapidly as possible. While it is always tempting to try to recover and clone all of the hybridomas producing specific antibody, cloning and freezing is the most time consuming aspect of the process. It is therefore useful to identify the most useful hybridomas as quickly as possible. For example, if high titers and high affinity antibodies are needed, the media from the initial hybridoma wells can be diluted 10 or even 100 fold and then screened. Many slower growing or low producing hybridomas will be missed but those can be identified by rescreening the original wells. With certain antigens, only high affinity antibodies will bind to ELISA plates containing a low density of antigen 3°. If antibodies of a certain class or subclass are needed, they can be screened directly by using antisera specific for that isotype. The instability of hybrids can be a problem but even very unstable hybridomas can be identified by cloning early and recloning repeatedly31. Instability can be reduced by using the system described by Taggart and Samloff32 in which the immunoglobulin heavy chain gene and the gene encoding resistance to a particular drug are on the same chromosome.
Conclusions
346
The quality and quantity of mouse and rat monoclonal antibodies can be improved by increasing immunogenicity, selecting the best animals, enriching for B cells making the desired antibody, increasing the efficiency of fusions, and screening judiciously. Additional improvement in each of these steps would be useful and may ultimately make it possible to recover the best and most useful antibodies which the individual is capable of producing. Such innovations may also make it possible to generate routinely human monoclonal antibodies which are still difficult to obtain and which would be very useful for diagnosis and therapy in vivo. In addition, there
would be many more uses of antigen binding sites if the structure of the antibody could be appropriately modified and this will be discussed in the second part of this article. Someof the work describedabovewas supportedby grantsfromthe NIH (AI105231)and NSF(DCB8316150).The HybridomaFacilityat Einsteinis supportedbya CancerCenterGrant(2P30CA13330).DeborahFrenchisaFellow of the NewYorkHeartAssociationandallof theauthorsarepartof theIrvington HouseInstitutefor MedicalResearch.Wewouldliketo thankDrJosephDaviefor hishelpfulsuggestions. References
1 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 2 Westerwoudt, R.J.(1985)J. ImmunoL Meth. 77, 181 196 3 Damjanov, I. and Knowles, B.B.(1983)Lab. Invest. 48, 510-525 4 Farr,R.S. and Dixon F.J.(1960)./.Immuno/. 85, 250-257 5 Weir, D.M. (ed.) (1973) Applications oflmrnunological Methods (2nd edn), Appendix 2, Blackwell Scientific
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