Monoclonal Antibodies Reveal the Structural Basis of Antibody Diversity Jean-Luc Teillaud, Catherine Desaymard, Angela M. Giusti Barbara Haseltine, Roberta R. Pollock, Dale E. Yelton Donald J. Zack, Matthew D. Scharff Immunologists have studied antibod- ies for two quite different reasons. On the one hand, antibodies are the final products of an immune response that evolved to protect vertebrates from an environment filled with a seemingly infi- patients or animals with this malignancy is a monoclonal antibody that is pro- duced by a transformed antibody-form- ing cell growing in an uncontrolled fash- ion. Much of what we know about the structure of antibody molecules and the Summary. Hybridoma technology has made it possible to introduce into continuous culture normal antibody-forming cells and to obtain large amounts of the immunoglob- ulin produced by each of these cells. Examination of the structure of a number of monoclonal antibodies that react with a single antigen has provided new information on the structural basis of the specificity and affinity of antibodies. Comparisons of families of monoclonal antibodies derived from a single germ line gene revealed the importance of somatic mutation in generating antibody diversity. Monoclonal antibod- ies that react with variable regions of other monoclonals allow the further dissection and modulation of the immune response. Finally, the continued somatic instability of immunoglobulin genes in cultured antibody-forming cells makes it possible to determine the rate of somatic mutation and to generate mutant monoclonal antibodies that may be more effective serological reagents. nite number of life-threatening infectious and toxic agents. An essential property of the immune response is its ability to generate enormous sequence diversity in antibody molecules: an individual can produce more than 108 different antibod- ies, each with a different amino acid sequence. To determine the genetic and molecular mechanisms responsible for this diversity, immunologists examine the primary structure of individual anti- body molecules and the genes encoding them. On the other hand, scientists in many areas have recognized the useful- ness of antibodies as reagents that can be used to identify, locate, and quantitate macromolecules in complex biological mixtures. However, the production of homogeneous antibodies that can be used as reagents for accurate and repro- ducible immunoassays has proved diffi- cult. The problem was to some extent solved when it was recognized that the disease multiple myeloma is a malignan- cy of antibody-forming cells. The large amount of paraprotein in the serum of 18 NOVEMBER 1983 organization and structure of the im- munoglobulin genes came from the anal- ysis of myeloma immunoglobulins and cells. However, only a few of these myeloma proteins were found to react with known antigens and it was not pos- sible to use myelomas to precisely dis- sect the enormous repertoire of antibod- ies or to harness the disease to produce homogeneous antibodies that would be useful serological reagents. Relatively large amounts of homogeneous antibod- ies could be produced by immunizing animals with some bacterial polysaccha- rides (1); small amounts of a wider varie- ty of antibodies could be obtained by limiting dilution cloning of antibody- forming cells in the spleens of irradiated animals and subsequent analysis of frag- ments of such spleens in short-term cul- ture (2). Both approaches provided im- portant information but were limited in their usefulness. A method for routinely producing large amounts of a wide variety of homo- geneous antibodies was discovered in 1975 by Kohler and Milstein (3). These workers were using cultured mouse my- eloma cells to study the regulation of immunoglobulin gene expression in so- matic cell hybrids. By fusing cultured mouse myeloma cells to normal spleen cells from immunized mice, they were able to introduce individual antibody- forming cells into long-term tissue cul- ture (3). Since immunization selectively increases the number of spleen cells pro- ducing antibody reactive with the immu- nizing antigen, a significant percentage of the hybrids, or hybridomas, were pro- ducing the desired antibody. Further- more, the clonal progeny of each hybrid- oma synthesized monoclonal antibodies all with the same amino acid sequence. The hybridomas retained the malignant properties of the myeloma parent, caus- ing tumors when injected into mice. The ascites fluid and serum of such tumor- bearing mice contained large amounts of the monoclonal antibody, making it pos- sible to obtain ten to hundreds of milli- grams of a desired antibody. The hybrid- oma cells could also be frozen and recov- ered at will and the exact same monoclo- nal antibody could be renewed when needed and was available indefinitely (3). Thus, Kohler and Milstein's discov- ery simultaneously satisfied the need for large amounts of chemically defined ho- mogeneous antibodies that were easily renewable for immunoassays and al- lowed the immunologists to repeatedly sample the repertoire of cells making antibody against a particular antigen and to study the protein and nucleic acid structure of representatives of this reper- toire. This has led to a better understand- ing of the genetic and molecular events responsible for antibody diversity. Molecular Basis of Antibody Diversity and Specificity Ever since it became obvious that anti- bodies specific for different antigens dif- fered from each other in the amino acid sequence of their variable (v) region (Fig. 1), there have been debates about whether each individual animal inherited in its germ line all of the genes required to code for the many antibody molecules they would produce during their lifetime, or if they inherited only a few germ line genes which subsequently underwent so- matic changes in their base sequence. Early studies by Weigert and Cohn and their colleagues on X light chains pro- duced by mouse myeloma cells suggest- ed that somatic mutation played a major The authors are members of the Department of Cell Biology at Albert Einstein College of Medicine, Bronx, New York 10461. 721