Am J Hum Genet 31:539 - 547, 1979

Monoclonal Antibodies. Hybrid Myelomas-A Revolution in Serology and Immunogenetics ROGER H. KENNETT'

INTRODUCTION

In 1975 Kohler and Milstein [1] reported an experimental method of obtaining hybrid plasmacytoma cell lines that make antibody against preselected antigens. Their approach was suggested by the. observation that hybrids made by fusing two antibody-producing cells continue to secrete the antibodies originally produced by each of the two parental cell types [2, 3]. They carried this observation one step further and stimulated what may well become a revolution in the fields of serology, immunology, and cell biology. They fused spleen cells, from a mouse immunized against sheep red blood cells, with P3X63Ag8, a plasmacytoma line. As many as 10% of the hybrid clones were found to secrete antibody against sheep red blood cells into the culture medium [1]. The frequency of hybrids making antibody against the recently injected antigen suggested a preferential fusion or hybrid formation with spleen cells recently stimulated by antigen. Kohler, Milstein and co-workers used the same procedure (fig. 1) to produce monoclonal antibodies against the hapten 2,4,6,-Trinitrophenyl, and mouse IgD [4, 5]. They also reported mouse plasmacytoma x rat spleen cell hybrids making antibody against a rat histocompatibility antigen [6] and leukocyte differentiation antigens [7]. It soon became clear that this technique of monoclonal antibody production is useful for producing antibodies against a wide range of antigens. Effectively, what one does in this procedure is to immunize (usually a mouse or rat) with an antigen containing an array of antigenic determinants, allow the animal to respond, and then, use the spleen cells to generate hybrid plasmacytomas. Each hybrid that secretes antibody to the antigen of interest is producing a single clonal component of the animal' s complex antibody response and is thus a so-called monoclonal antibody. Preliminary to the production of hybrids making antibody against a chosen antigen, it is essential that there be available an assay procedure that enables the discrimination Received April 16, 1979. The author is supported by grants CA-24263, CA-18930, and GM-20138 from the National Institutes of Health, and grant PCM76-82997 from the National Science Foundation. I Department of Human Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19174. © 1979 by the American Society of Human Genetics. 0002-9297/79/3105-0012$01.00

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HYBRIDOMA PRODUCTION 3DAYS SPLEEN

IMMUNIZE

IMMUNIZE

10" SPLEEN CELLS ROC REMOVED

CLONES PRODUCING ANTIBODY FROM SPLEEN GENOME

HYBRID MYELOMA SELECTED IN HAT

FUSION WITH PEG

1I7 MOUSE PLASMACYTOMA HGPRT

FIG. 1. -Spleen cells from an immunized mouse are fused with a mouse plasmacytoma line. Polyethylene glycol or Sendai virus is used as a fusing agent. Fused cells are plated into several wells and grown in HAT (hypoxanthine, aminopterin, thymidine) medium. The cell line lacking the enzyme HGPRT will not grow in this selective medium. Spleen cells have a limited lifespan in culture and do not form visible colonies. Hybrid cells grow to form visible colonies in 2-3 weeks. Supernatants are tested for antibody, and those hybrids making antibody with the desired specificity are grown and recloned. Monoclonal antibody can then be obtained from the supernatant of these hybrid cells. Cells can also be injected into a mouse and ascites fluid or serum containing the antibody in mg/ml amounts obtained from the mouse. (Figure by Kendra Eager.)

between antibodies reacting with that antigen, and those reacting with other antigens to which the mouse has been exposed. This means that in the case of hybridoma antibody production, a crude antigen mixture may be used for immunization as long as the assay procedure used allows you to select the specific antibody you want from those produced by a collection of hybridomas. A variety of assays may be used, depending on the properties of the antigen [8]. To screen for antibodies against cell surface antigens, one can use complement mediated cytotoxicity which will detect those classes of antibody which bind complement. In some cases, for instance in the case of soluble antigen, a more general binding assay is useful. To test for antibody binding to the chosen antigen, one uses that antigen in some solid phase form. The hybridoma supernatants are incubated with cell pellets or antigen attached to tubes, plates, or filter paper discs. The antigen is washed, and the amount of hybridoma immunoglobulin attached to the antigen is determined with a second reagent such as rabbit anti-mouse immunoglobulin labeled with 125I Bound antibody may also be detected by [1251] Staphylococcus aureus protein A, a reagent that binds to certain classes of immunoglobulin better than others. The design of the screening assay should be based on the intended use of the antibody. For example, whether you need an antibody that is cytolytic, or one that can be used to precipitate a

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solubilized antigen by reacting with protein A-bearing S. aureus, will affect which assay you use to screen supernatants for activity. Using the appropriate screening procedure, one may test the supernatant of several hundred hybrid clones at an early stage and select positive cultures for expansion as sources of desired antibody. A hybrid culture supernatant is capable of producing clonal antibody in quantities on the order of 100 tkg/ml, and when the hybrid is injected into a mouse as a tumor, ascites fluid or serum containing the antibody in mg/ml amounts can be obtained. The hybridoma system thus provides a large supply of specific antibody which is in continuous supply and can be produced without absorption of unwanted antibodies. The same reagents can then be distributed to various laboratories, so that results in different experimental systems can be more readily compared. These reagents can be used in procedures for which antisera were previously used but with more confidence that a single antigenic determinant is being identified, and that the antigen detected in different laboratories using different methods is actually the same. This approach has allowed the isolation of mouse antibodies that recognize human polymorphic differences [9, 10], and antibodies directed against specific human cell types [10-12]. As a part of a mouse's total serum antibody response, each of these antibodies would be only a minor component in the total set of antibodies stimulated by the human antigens recognized as foreign by the mouse. The hybridoma system allows individual components of a complex response to be separated from each other and to be produced in large quantities. Researchers in many areas of the biological sciences have recognized the implications of Kohler and Milstein's results and are making monoclonal antibodies against a wide range of antigens [8,12. This symposium volume details methods, and the preface includes an extensive list of hybridomas produced in several laboratories]. The following discussion is not intended as an exhaustive review of the available literature on this subject but is intended to provide an overview of many of the applications of monoclonal antibodies to genetics and cell biology. Monoclonal Antibodies as Tools for the Study ofHuman Genetics

Obtaining specific antisera against human cell surface antigens or against human polymorphic differences has been more difficult than in other species where one may routinely immunize an animal with cells or macromolecules from another animal of the same species. Often extensive absorptions of antisera are needed to remove general antihuman antibodies to produce an antiserum that has the desired specificity. The production of monoclonal antibodies against human antigens makes it possible to obtain antibodies that react only with a single selected antigen, and in fact, with a single antigenic determinant on that antigen. Monoclonal antibodies have been made against several human cell surface antigens including leukocyte differentiation antigens [8, 11], tumor associated antigens [1315], general human species antigens [9], a blood group specificity [9], and a polymorphic human histocompatibility antigen (HLA) determinant [10]. By fusing spleen cells from a mouse injected three times with purified plasma membrane from

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human tonsil cells, Barnstable et al. [9] obtained hybrids making antibodies to group A erythrocytes, a human species antigen previously mapped on chromosome 11, and an antibody reacting with the 43,000 dalton molecular weight polypeptide of the HLA-A, B, and C loci. Bodmer and co-workers used this same monoclonal antibody to prepare an affinity column on which the A, B, and C HLA chains were purified [ 16]. From a mouse immunized with purified HLA-A2, Parham and Bodmer obtained a hybridoma making monoclonal antibody against the HLA-A2 allotypic specificity [ 10]. Anti-HLA antibodies made in this way have many advantages over the HLA antisera usually obtained from multiparous women. Usually such sera are of relatively low titer and contain antibodies against more than one HLA specificity, thus HLA typing is usually done with a large panel of sera and the HLA type determined by the pattern of reactivities of the sera panel against a person's lymphocytes. Monoclonal antibodies thus provide a source of very useful reagents for HLA typing and potentially for detecting other human antigenic polymorphisms. Monoclonal antibodies have been made against human melanoma-associated antigens [15], and a human oncofetal antigen present on neuroblastomas [13], as well as antibodies against subpopulations of human lymphocytes [8, 11, 12]. These antibodies will make it possible to isolate and characterize these antigens and also to identify and separate subpopulations of cells bearing these antigens. Such reagents are also useful for detecting the expression of these antigens on mouse x human hybrids and thus determination of the chromosomal location of the loci producing these molecules [9]. It has been found recently that monoclonal antibodies reacting with polymorphic specificities on products of the rat or mouse major histocompatibility complex (MHC) also detect polymorphic differences in human ([17], and T. McKearn, personal communication, 1979). This not only prompts interesting hypotheses concerning how such polymorphic differences have been maintained in several species, but also provides a source of reagents for detecting human polymorphic differences. Several investigators are directing efforts toward the production of hybridoma antibodies against enzymes or other soluble proteins. Our laboratories have made antibodies against human alkaline phosphatase including an antibody that reacts with two common allelic forms of the enzyme but shows no reaction with another common allelic form (Slaughter et al., unpublished results). These results and other reports of antibodies against human allotypic specificities such as type A erythrocytes and HLA-A2 indicate that it will not be difficult to obtain monoclonal antibodies that allow useful discriminations between allelic and isotypic forms of human gene products. Mouse x human hybrid plasmacytomas making human immunoglobulins [14, 18] make it possible to obtain large amounts of an assortment of human immunoglobulin chains. This may contribute to the study of human immunoglobulin idiotype and variable (V) region genetics, and thus do what the introduction of procedures for induction of mouse plasmacytomas did for mouse Ig genetics [ 19]. The availability of a larger assortment of monoclonal human Ig chains will make it easier to produce monoclonal antibodies against human idiotypic (antigenic specificities present on the V region of an antibody and therefore useful for detection of the expression of specific V region genes), isotypic, and allelic Ig differences. It is therefore likely that such markers will soon be more ea6ily studied in families and populations.

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Monoclonal Antibodies and the Analysis of Mouse Ig Genetics The availability of inbred strains of mice, development of systems for analyzing the murine B-cell response in vitro, and the development of techniques for analyzing DNA sequences have contributed to the significant progress in understanding the genetics of immunoglobulins. Several laboratories have used N. Klinman's spleen fragment technique to analyze the development of the B-cell repertoire in the neonatal stages of mouse development [20, 21]. Studies with this technique have shown that during the first few days after birth, a limited number of clones make antibody against a particular hapten such as 2,4-Dinitrophenyl. The early clonotypes that appear in a particular strain of mice against a particular hapten are consistently of the same class and isoelectric point. During this neonatal period, diversification of the response takes place until many types of antibodies against the same determinant are produced. These differ in both region structure and Ig class. The clones of cells detected within spleen fragments are limited in their usefulness, because they survive for a few weeks at most and thus produce limited amounts of antibody. This makes it impractical to compare the structure of the neonatal antibody to that which is made at larger stages in the ontogeny of the immune response. Hybridomas have made it possible to "capture" the products of the neonatal clones, as well as clones from later developmental stages, as continuous hybrid cell lines [8]. The antibodies secreted by the cell lines can be collected in large amounts for sequence analysis, and the DNA specific for the immunoglobulin genes can be isolated and studied [22]. The information obtained by these analyses leads to a better understanding of the genetic mechanisms of antibody diversification, which may have implications for other aspects of the control of gene expression and differentiation. The ability to obtain several monoclonal antibodies against specific haptens also makes it possible to use these antibodies to develop anti-idiotypic reagents. These reagents are antibodies directed against antigenic determinants on the region of specific immunoglobulin molecules, and provide a way of detecting V region genes in a specific mouse or group of mice. Before hybridomas, one could, with much work, obtain anti-idiotypic reagents against certain predominant idiotypes. Hybridomas provide a way of analyzing a large number of different Ig V regions and will make possible a more thorough analysis of mouse V region genetics.

Monoclonal Antibodies and the Analysis of Rodent Cell Surface Antigens Understanding the genetics of antigens in the MHC of vertebrates has been facilitated by inbred strains of mice and rats. These genetically homogeneous strains have made it possible to produce antisera against a restricted number of antigenic differences that exist between one strain and another. The antisera obtained when cells from one strain of mice are injected into another are complex and contain antibodies against a variety of MHC antigens, and often react with many different specificities on a given molecule. Hybridomas have made it possible to obtain monoclonal antibodies that detect a single antigenic difference [6, 7]. Because of the clonal nature of hybridoma antibodies, they do not correspond to just those antibodies that compose the major component of antisera. They may be

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antibodies against antigenic determinants on cells that would not normally be detected with antisera because the antigens are on a small fraction of the cell population, or because the antigen is less immunogenic or is quantitatively insignificant among the major antigens of the cell surface. Williams et al. [7] reported several hybridoma lines making antibodies against subpopulations of rat lymphocytes which had not been detected by standard antisera. McKearn et al. [24] have made monoclonal antibodies against rat histocompatibility antigens and have shown that they have the same in vivo effect as antisera against the rat MHC antigens (AgB antigens). As has been demonstrated with antisera, injection of these antibodies suppresses the response of an incompatible recipient that has been injected with cells of the histocompatibility type detected by the antibody. This not only confirms that the antibodies in the serum that were effective in suppressing the response were those against the AgB antigens, but shows that monoclonal antibodies will be as useful as sera for in vitro functional studies, and may even have potential therapeutic value. Bechtol et al. [25] have obtained antibodies against mouse testis specific antigens which were stimulated by syngeneic immunization of testis, because the developing testis cells are normally sequestered from the immune system. Thus antigens which are testis specific are considered foreign when injected into the circulation of mice of the same strain. This makes it possible to obtain antibodies against antigens which are on a small number of differentiating cells within a mixture, and which in a standard antisera would probably be obscured in a collection of antibodies against a variety of antigenic differences. Monoclonal Antibodies Against Microorganisms Koprowski et al. reported the production of monoclonal antibodies against influenza, parainfluenza, rabies, SV40, and herpes viruses [14, 26]. Such antibodies appear to have a degree of specificity that permits an analysis of the structure and genetic variation of specific viruses. Previously single antigenic determinants were usually defined by the pattern of reactions between a set of antiviral sera and a panel of different virus strains. Now that the presence of a specific determinant on a given virus type can be detected, it is possible to screen more easily among the progeny of a given viral strain for antigenic variants. Such reagents are potentially useful for clinical detection and classification of bacteria as well as viruses. CONCLUDING REMARKS

Since the first hybridomas were made in 1975, the technology of producing and analyzing the monoclonal antibodies has developed rapidly. Currently there are variant myeloma lines which do not produce any immunoglobulin of their own but when fused to spleen cells, form hybrids which synthesize the antibody produced by the spleen cell genome. This eliminates the possibility of hybrid molecules formed from the immunoglobulin chains produced by the myeloma genome and the spleen cell genome [27, 28]. Monoclonal antibodies have been used to prepare immunoabsorbent columns [16] for isolation of antigens and have been used to label cells to be separated by the fluorescent activated cell sorter [29]. It is clear that monoclonal antibodies can be

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applied to most of the techniques for which standard antisera have been so useful, with the added advantage that large quantities of pure antibody may be easily obtained. With proper monitoring of the activity and specificity, one can be sure that the same antibody is constantly available and can be used in several laboratories. Another advantage is that antibodies synthesized in culture can be labeled internally with radioactive amino acids. Bottcher et al. recently reported the isolation of a hybridoma making monoclonal IgE [30]. From this line they can obtain large quantities of this immunoglobulin class which is found in very low serum concentrations. This illustrates an additional advantage of using somatic cell hybrids as a source of biologically interesting products of specialized cells. By using the proper combination of cells, this can perhaps be applied to the isolation of other interesting molecules such as hormones or T-cell factors [31]. There are several factors that still remain to be defined and developed in applying monoclonal antibodies to genetics ,and cell biology. Considering the variants that can arise in the Ig produced by myeloma cell lines [32], ways of monitoring the specificity of the antibodies used as standard typing reagents will have to be defined. The concept of "antigenic determinant" may have to be more clearly defined now that specific probes for such entities exist. One may find that an antibody directed against a determinant on the molecule used as an immunogen may also appear on other molecules. For example, considering the homology between human 2 microglobulin and IgG [33, 34], mouse antibodies against /2 microglobulin may also react with IgG. This possibility makes it necessary to carefully characterize the antigens with which a given antibody reacts. At the same time this presents increasing possibilities for those interested in the antigenic relationships between different proteins. When one considers the implications of such an increased degree of specificity and the large amounts of monoclonal antibody that can be produced, it is clear that exciting and stimulating observations will be derived from the application of monoclonal antibodies to many areas of biology. REFERENCES 1. KOHLER G, MILSTEIN C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497, 1975 2. COTTON RGH, MILSTEIN C: Fusion of two immunoglobulin in-producing myeloma cells. Nature 244:42-43, 1973 3. SCHWABER J, COHEN EP: Pattern of immunoglobulin synthesis and assembly in a human-mouse somatic cell hybrid clone. Proc Natl Acad Sci USA 71:2203-2207, 1974 4. KOHLER GU MILSTEIN C: Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol 6:511 - 519, 1976 5. PEARSON T, GALFRi G, ZIEGLER A, MILSTEIN C: A myeloma hybrid producing antibody specific for an allotypic determinant on "IgD-like" molecules of the mouse. Eur J Immunol 7:684-690, 1977 6. GALFRi G, HOWE SC, MILSTEIN C, BUTCHER GW, HOWARD JC: Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:550-552, 1977 7. WILLIAMs AF, GALFRi G, MILSTEIN C: Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes. Cell 12:663-673, 1977 8. KENNETT RH, DENIS KA, TUNG AS, KLINMAN NR: Hybrid plasmacytoma production: fusions with adult spleen cells, monoclonal spleen fragments, neonatal spleen cells and

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human spleen cells. Curr Top Microbiol Immunol 81:77-91, 1978 9. BARNSTABLE CJ, BODMER WF, BROWN G, et al.: Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis. Cell 14:9-20, 1978 10. PARHAM P, BODMER WF: Monoclonal antibody to a human histocompatibility alloantigen HLA-A2. Nature 276:397- 399, 1978 11. TRUCCO MM, STOCKER JW, CEPPELLINI R: Monoclonal antibodies against human lymphocyte antigens. Nature 273:666-668, 1978 12. LEVY R, DILLEY J, LAMPSON LA: Human normal and leukemia cell surface antigens. Mouse monoclonal antibodies as probes. Curr Top Microbiol Immunol 81:164-169, 1978 13. KENNETT RH, GILBERT F: Hybrid myelomas producing antibodies against a human neuroblastoma antigen present on fetal brain. Science 203:1120-1121, 1979 14. KOPROWSKI H, GERHARD W, WIKTOR T, MARTINIS J, SHANDER M, CROCE CM: Anti-viral and anti-tumor antibodies produced by somatic cell hybrids. Curr Top Microbiol Immunol 81:8-19, 1978 15. KoPRoWSKI H, STEPLEWSKI Z, HERLYN D, HERLYN M: Study of antibodies against human melanoma produced by somatic cell hybrids. Proc Natl Acad Sci USA 75:3405-3409, 1978 16. PARHAM P. BARNSTABLE CJ, BODMER WF: Properties of an anti-HLA-A-B-C monoclonal antibody. J Immunol. In press, 1979 17. GASSER DL, WINTERS BA, McKEARN TJ, KENNETT RH: Monoclonal antibody directed to a B cell antigen present in rats, mice, and humans. Proc NatlAcad Sci USA. In press, 1979 18. LEVY R, DILLEY JS, KORA K, KUCHERLAPATI R: Mouse-human hybridomas. The conversion of non-secreting human B cells into Ig secretors. Curr Top Microbiol Immunol 81:170-175, 1978 19. POTTER M, CANCRO M: Plasmacytogenesis and the differentiation of immunoglobulinproducing cells, in Cell Differentiation and Neoplasia, edited by SAUNDERS GF, New York, Raven Press, 1978, pp 145-161 20. KLINMAN NR, SIGAL WH, METCALF ES, PIERCE SK, GEARHART PJ: The interplay of evolution and environment in B-cell diversification. Cold Spring Harbor Symp Quant Biol 41:165-173, 1977 21. KLINMAN NR, METCALF ES, SIGAL NH: The functional analysis of B cell maturation at the clonotype level, in Development of Host Defenses, edited by DAYTON D, New York, Academic Press, 1976, pp 93-106 22. TONEGAWA S, BRACK C, HozoMI N, MATTHYSSENS G, SCHULLER R: Dynamics of immunoglobulin genes. Immunol Rev 36:73-94, 1977 23. Oi VT, JONES PP, GODING JW, HERZENBERG LA, HERZENBERG LA: Properties of monoclonal antibodies to mouse Ig allotypes H-2 and Ia antigens. Curr Top Microbiol Immunol 81:115- 129, 1978 24. McKEARN T, ARMIENTO M, WEISS A, STUART FP, FITCH FW: Selective suppression of reactivity to rat histocompatibility antigens by hybridoma antibodies. Curr Top Microbiol Immunol 81:61-65, 1978 25. BECHTOL K, BROWN S, KENNETT RH: Recognition of differentiation antigens of spermatogenesis in the mouse by using antibodies from spleen cell-myeloma hybrids after syngeneic immunization. Proc Natl Acad Sci USA 76:363-367, 1979 26. KOPROWSKI H, GERHARD W, CROCE CM: Production of antibodies against influenza virus by somatic cell hybrids between mouse myeloma and primed spleen cells. Proc Natl Acad Sci USA 74:2985-2988, 1977 27. KOHLER G, HENGARTHER H, SHULMAN MJ: Immunoglobulin production by lymphocyte hybridomas. Eur J Immunol 8:82-93, 1978 28. SHULMAN M, WILDE CD, KOHLER G: A better cell line for making hybridomas secreting specific antibodies. Nature 276:269-270, 1978 29. WHITE RAH, MASON DW, WILLIAMS AF, GALFRi G, MILSTEIN C: T-lymphocyte heterogeneity in the rat: separation of functional subpopulations using a monoclonal antibody. J Exp Med 148:664-673, 1978

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30. BOTTCHER I, HAMMERLING G, KAPP JF: Continuous production of monoclonal mouse IgE antibodies with known allergenic specificity by a hybrid cell line. Nature 275:761-762, 1978 31. SIMPSON E, KONTIAINEN S, HERZENBERG LA: T cell hybrids will T cell functions. Curr Top Microbiol Immunol 81:195-202, 1978 32. FRANCUS T, DHARMGRONGARTAMA B, CAMPBELL R, SCHARFF MD, BIRSHTEIN B: Ig62.-producing variants of an IgG2b-producing mouse myeloma cell line. J Exp Med 147:1535-1550, 1978 33. SMITHIES 0, POULIK MD: Initiation of protein synthesis at an unusual position in an immunoglobulin gene. Science 175:187-189, 1972 34. PETERSON PA, CUNNINGHAM BA, BERGGARD I, EDELMAN GM: f32-microglobulin-a free immunoglobulin domain. Proc Natl Acad Sci USA 69:1697- 1701, 1972