Antibody Expression and Engineering

Preface molecules of intense interest to the biotechnology community. Antibodies are immunoglobulin molecules of the immune system developed by higher organisms to combat the invasion of foreign substances (antigens). Each antibodty has specific activity for the foreign material that elicits its synthesis. In nature, each antibody is made by a different line of lymphocytes and their derived plasma cells in a specific animal. If one could pick out such a cell making a single specific antibody and grow it in a culture, then the cell's progeny, or clone, would be a source of large amounts of identical antibody against a single antigenic determinant: a monoclonal antibody (MAb*). Unfortunately, antibody-secreting cells cannot be maintained well in a culture medium. In 1975, Kohler and Milstein fused mouse myeloma cells with lymphocytes from the spleen of mice immunized with a particular antigen. The resulting hybrid myeloma, or "hybridoma," cells expressed both the lymphocytes' property of specific-antibody production and the immortal character of myeloma cells. Since then the hybridoma technology has been widely adopted as a method of choice for the preparation of MAbs to a wide spectrum of antigens. Various hybridomas can be cloned and grown in large quantity for indefinite periods of time, and they secrete high concentrations of monoclonal antibodies. Depending on the source of lymphocyte and myeloma cells and by using additional powerful genetic manipulation techniques, different combinations of hybrids (mousemouse, mouse-human, and human-human hybridomas) have now been synthesized. Some of the real or potential applications of monoclonal antibodies will require kilogram quantities of highly purified antibody. Therefore, cost-efficient methods for producing large quantities of highly purified monoclonal antibodies must be developed. Process development will be an important factor for the successful commercialization of any of the therapeutic monoclonal antibodies. Through better understanding of the nutritional requirements of these cells, many improvements in culture media and feeding strategies have been developed. Costly and undefined serum components have been eliminated from media formulation without

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ANTIBODIES ARE MULTTDOMAIN GLYCOPROTEIN

* A number of abbreviations for monoclonal antibodies are used in this book: MAb, Mab, Abs, MoAbs. They all denote the same thing.

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Wang and Imanaka; Antibody Expression and Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


impairing the immunoglobulin secretion or the consistency of the anti body properties. Development of serum-free media has also made the antibody-manufacturing process more economically competitive. Many antibodies have the potential to be the most economically signi ficant products derived from mammalian cells. They are already being used extensively in diagnostic assays and their therapeutic potential is increasing with the development of affinity purification systems and in vivo imaging. The immunotherapeutic application uses monoclonal anti bodies either as so-called "magic bullets" to destroy malignant tissue (cancer therapy) or as target-specific drug delivery agents (for immuno logical diseases). Recent progress in our ability to genetically manipulate and alter the antibody molecules and then express the whole antibody molecule or its selected fragments in various recombinant hosts has profoundly altered the fields of protein chemistry and molecular immunology. About the Book In the first few chapters of this book, recent advances in antibody expres sion and production of several industrially important monoclonal antibo dies based on traditional myeloma cells are described. Using a case-study approach, David Robinson and his colleagues show how gene amplifica tion, medium optimization, and process development can be used to optimize a humanized anti-CD18 antibody. Almost 1 g/L of monoclonal antibody can be routinely produced in an optimized fed-batch culture. Sawada and Kitano describe the development and characterization of a human monoclonal antibody against hepatitis Β virus surface antigen (HBsAg). Humanization of the MAbs is now routinely carried out to design therapeutics that minimize the formation of human anti-mouse antibodies ( H A M A ) for various in vivo applications. Besides using myeloma cells as hosts, M . E . Reff s group has developed an efficient expression system to produce whole immunoglobulin molecules in Chinese hamster ovary (CHO) cells. Almost 800 mg/L of antibody was achieved in a 100-L fermentor in 6 days. This is quite competitive with the traditional myeloma hosts. Genetic shuffling of antibody domains and other active molecules can also be used to create new chimeric molecules with various combinations of binding and effector functions and novel antigen-binding molecules. Similarly, recombinant D N A techniques have now been used to produce different antibody fragments with reduced molecular mass, such as Fab, Fv, or single-chain antigen-binding molecules. These engineered mono clonal antibodies and their corresponding fragments have the potential to expand on various clinical applications, such as diagnostic imaging and immunotherapeutics, that require smaller molecules for deeper penetra tion or with altered binding properties. The next few chapters are devoted χ Wang and Imanaka; Antibody Expression and Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


to the expression of monoclonal antibody and its fragments in other nonmammalian host systems. Potter et al. demonstrate the use of baculovirus expression system to produce various forms of hybrid or chimeric antibodies of IgG, IgM, and IgA isotypes in insect cells. Julian M a reports on the progress of expressing antibody fragments in cultivated plant cells and plants. Microbial gene expression systems have also been used routinely to produce antibody fragments (such as Fab and Fv) consisting of one or two antibody chains. Co-expressing antibody light and heavy chains in various microbial hosts have been demonstrated since 1984. David F i l pula and his colleagues review the potential of using the Escherichia coli expression system to overproduce a single-chain Fv protein with human IgM Qx specificity. Better and Nolan describe the construction of a family of immunofusion proteins linking various antibody fragments of the T-cell targeted H65 antibody with the plant ribosome-inactivating protein called gelonin in E. coli. Some of these fusion proteins are as potent as some corresponding chemical conjugates developed for use as immunotoxins. S. L . Wong and his group have shown that a multiple-proteasedeficient Gram-positive Bacillus subtilis can be used as an alternate expression host to produce an anti-digoxin single-chain antibody fragment. Nyyssonen and Keranen discuss the use of cellulase-producing Trichoderma reesei as a promising host to produce a Fab fragment fused to the cellulase gene. In the remaining chapters, Lavey and Janda review the exciting and promising field of catalytic antibodies and their applications in organic synthesis. For a more specific application, T. Imanaka's group have successfully generated a light chain of a M A b against porphyrin that still retains peroxidase-like activity. This book covers many recent developments in antibody expression and engineering using traditional hybridoma and nontraditional recombinant host systems and a few potential applications. It is not intended to be a comprehensive handbook. Increasingly, fine details of the antibody molecule have become known through X-ray crystallography and N M R spectroscopy. Thus, quantitative relationships between structure and function can now be estimated and manipulated through genetic engineering. Similarly, screening for novel antibodies through the generation of combinatorial libraries using filamentous phage and other microbial antibody expression systems is becoming routine. Therefore, the advances in genetic manipulation and process development discussed in this book coupled with other advances will propel the field of antibody engineering to a new horizon. Acknowledgments We gratefully acknowledge the assistance of many anonymous reviewers xi Wang and Imanaka; Antibody Expression and Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

who helped us carry out in-depth reviews and supplied important and constructive criticism of the original manuscripts. We also acknowledge the patience and support from the staff of the A C S Books Department, particularly Barbara Pralle, that made this book a reality. H E N R Y Y. W A N G

Department of Chemical Engineering University of Michigan Ann Arbor, MI 48109-2136


TADAYUKI IMANAKA

Department of Biotechnology Faculty of Engineering Osaka University Yamadaoka, Suita Osaka 565, Japan June 14, 1995

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Wang and Imanaka; Antibody Expression and Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

Antibody Expression and Engineering

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