27 The Israel Chemist and Chemical Engineer Issue 9 · January 2023 · Tevet 5783 History of Chemistry Articles of four polypeptide chains, two identical light chains and two identical heavy chains, and can be thought of as forming a flexible Y-shaped structure. Antibodies produced by myeloma cells are all identical. At this time Georges Köhler joined the group as a post-doc. His project was to grow a myeloma cell in culture capable of recognizing an antigen, in order to derive mutants with altered affinity for the antigen. The project was not successful. Milstein: “[Köhler] started a side project that involved variations on the theme of hybridization of two myeloma cells. The combination of the need of antibody-producing cells in tissue culture, which could not grow, and the experiments with hybrid myelomas, did the trick. Why not try to make the cell we needed? Perhaps we could substitute one myeloma cell for an antibody producing normal B-cell? Normal antibodyproducing B cells die very quickly, but perhaps we could immortalize the antibody production by fusion with the myeloma cell line? To our surprise, the experiments were a resounding success from start to finish. Within a short time, we derived cell lines in culture making antibodies against preselected antigen.” [15-16] Preparing cell lines against a pre-selected antigen The steps in the production of cell lines against a preselected antigen are summarized on the Nobel Prize website for 1984: “Spleen cells are prepared from animals, usually mice, which have been immunized with a selected antigen. These cells are then fused with myeloma cells maintained in culture in the laboratory. The product of this fusion is referred to as a hybridoma. Surprisingly, a hybrid of two cells can survive and also continue to divide. In this particular hybrid the myeloma cells contribute the capacity for survival, whereas the spleen cells direct the synthesis of antibodies with the preselected specificity. By special arrangements it is possible to achieve a multiplication of hybridoma cells but not of isolated myeloma cells. The hybrids obtained are propagated in a highly diluted state so that colonies deriving from single hybrid cells can be isolated. By use of a sensitive method the clones which produce the specific antibodies are identified. A particular hybridoma can then be used for future, unlimited production of a highly specific antibody.” [6] Köhler and Milstein: “We used sheep red blood cells (SRBC) as immunogen (An immunogen is a specific type of antigen that is able to elicit an immune response.), which enabled us, after culturing the fused lines, to determine the presence of specific antibody producing cells by a plaque technique. The hybrid cells were cloned in soft agar and clones producing antibody were easily detected by an overlay of SRBC and complement. Individual clones were isolated and shown to retain their phenotype as almost all the clones of the derived purified line are capable of lysing SRBC. The clones were visible to the naked eye.” [1] Successful results Köhler and Milstein: “Three different experiments were successful in producing a large number of antibody-producing cells. Three weeks after the initial fusion, 33/1,086 clones (3%) were positive by direct plaque assay. The cloning efficiency in the experiment was 50%. In another experiment, however, the proportion of positive clones was considerably lower (about 0.2%). In a third experiment the hybrid population was studied by limiting dilution analysis. From 157 independent hybrids, as many as 15 had anti-SRBC activity. The proportion of positive over negative clones is remarkably high. It is possible that spleen cells which have been triggered during immunization are particularly successful in giving rise to viable hybrids. It remains to be seen whether similar results can be obtained using other antigens. (The technology was soon extended to every conceivable immunogen [7] .) “The cells used in this study are all of BALB/c (laboratorybred strain of the house mouse) origin and the hybrid clones can be injected into BALB/c mice to produce solid tumors and serum having anti-SRBC activity. It is possible to hybridise antibody-producing cells from different origins. Such cells can be grown in vitro in massive cultures to provide specific antibody. Such cultures could be valuable for medical and industrial use.” [1] The hybridoma revolution: an offshoot of basic research Milstein, in the introduction to the 1999 paper called “The hybridoma revolution: an offshoot of basic research,” wrote, “The production of monoclonal antibodies against predefined and, even more importantly, novel antigens has had an enormous impact in biology, medicine, and industry. Indeed, the hybridoma technique has been one of the pillars of the biotechnology revolution. Yet, none of the current applications were the goal of the research that made it possible. With hindsight, it may seem obvious that the invention of a method to immortalize cells that produce specific antibodies should have such potential. At the time, however, these most important applications were neither in our minds nor in the minds of biologists or even immunologists. When we stated in the original study that ‘Such (monoclonal antibody) cultures could be valuable for medical and industrial use’ we were thinking about immunoassays and passive therapy. It was only later that we started to consider seriously other possibilities. The technology was based on methods of somatic cell genetics, which we were using to analyse the origin of
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