This problem was solved by Georges Köhler and César Milstein, who devised a technique for producing a homogeneous population of antibodies of known antigenic specificity. They did this by fusing spleen cells from an immunized mouse to cells of a mouse myeloma to produce hybrid cells that both proliferated indefinitely and secreted antibody specific for the antigen used to immunize the spleen cell donor. The spleen cell provides the ability to make specific antibody, while the myeloma cell provides the ability to grow indefinitely in culture and secrete immunoglobulin continuously. By using a myeloma cell partner that produces no antibody proteins itself, the antibody produced by the hybrid cells comes only from the immune spleen cell partner. After fusion, the hybrid cells are selected using drugs that kill the myeloma parental cell, while the unfused parental spleen cells have a limited life-span and soon die, so that only hybrid myeloma cell lines, or hybridomas, survive. Those hybridomas producing antibody of the desired specificity are then identified and cloned by regrowing the cultures from single cells (Fig. A.9). Because each hybridoma is a clone derived from fusion with a single B cell, all the antibody molecules it produces are identical in structure, including their antigen-binding site and isotype. Such antibodies are called monoclonal antibodies. This technology has revolutionized the use of antibodies by providing a limitless supply of antibody of a single and known specificity. Monoclonal antibodies are now used in most serological assays, as diagnostic probes, and as therapeutic agents. So far, however, only mouse monoclonals are routinely produced in this way, and efforts to use the same approach to make human monoclonal antibodies have met with limited success. ‘Fully human’ therapeutic monoclonal antibodies are currently made by using phage display technology (described in Section A-8), by using recombinant DNA technology to clone and express antibody genes from human plasma cells (see Section A-9), or by immunizing transgenic mice (see Section A-34) carrying human antibody genes.
A-8 Phage display libraries for antibody V-region production.
In a technique for producing antibody-like molecules, gene segments encoding the antigen-binding variable, or V, domains of antibodies are fused to genes encoding the coat protein of a bacteriophage. Bacteriophages
Construct fusion protein of V region with a bacteriophage coat protein Cloning a random population of variable regions gives rise to a mixture of bacteriophages— a phage display library Select phages with desired V regions by specifc binding to antigen VH antibody protein DNA Isolate population of genes encoding antibody variable regions VH VL VL
Fig. A.10 The production of antibodies by genetic engineering.
Short primers to consensus sequences in heavyand light-chain variable (V) regions of immunoglobulin genes are used to generate a library of heavyand light-chain V-region DNAs by PCR, with spleen DNA as the starting material. These heavyand light-chain V-region genes are cloned randomly into filamentous phages such that each phage expresses one heavy-chain and one light-chain V region as a surface fusion protein with antibody-like properties. The resulting phage display library is multiplied in bacteria, and the phages are then bound to a surface coated with antigen. The unbound phages are washed away; the bound phages are recovered, multiplied in bacteria, and again bound to antigen. After a few cycles, only specific high-affinity antigen-binding phages are left. These can be used like antibody molecules, or their V genes can be recovered and engineered into antibody genes to produce genetically engineered antibody molecules (not shown). This technology may replace the hybridoma technology for producing monoclonal antibodies, and has the advantage that humans can be used as the source of DNA.