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Alternatives to Monoclonal Antibody Production (Proceedings)

Overview of the Science and Uses of Monoclonal Antibodies

Mark J. Soloski, PhD
Department of Medicine
 Johns Hopkins University School of Medicine
Baltimore, MD

Following challenge with foreign substances (antigens), the immune system of vertebrates can recognize and respond with the production of high affinity binding proteins (antibodies) that can interact with and clear the antigen. This property has been known for more than a hundred years and was a major conceptual advance in the understanding of protective immunity. In addition, this property has lead investigators to utilize the immune system in the generation of reagents that have been employed in a variety of experimental and applied scientific settings.

The feature of the immune system that is central to this capability is the evolution of a unique genetic mechanism that allows an individual to generate an almost limitless number of antibodies capable of reacting with the universe of foreign antigens. This process involves the ordered recombination of mini-gene segments so that a single lymphocyte exclusively expresses one antibody species. Thus, within an individual, which contains millions of lymphocytes, millions of distinct clonally expressed receptors are present. When antigen is introduced into a host, those lymphocytes that expressing antibody receptors that bind the antigen are selected and driven to produce soluble antibody. In this manner serum concentrations of antibodies that can bind antigen increases markedly following immunization, with the result being the generation of a polyclonal reagent with selective reactivity toward the foreign antigen. This approach has been utilized to generate highly specific reagents toward proteins, lipids, and carbohydrates as well as synthetic compounds.

In the 1970's Kohler and Milstein utilized somatic cell hybridization to develop an approach to immortalize individual antibody producing cells. As a result there is now the capability of generating cell lines that would produce a single antibody species (a monoclonal antibody). This approach offered a number of advantages over the use of serum from immunized hosts. A standardized procedure could be adapted that provided for an unlimited source of the monoclonal antibody, eliminated the need for highly purified antigen for immunization, and allowed the investigator to design immunization and screening strategies to generate highly specific monoclonal reagents toward antigens of scientific interest.

Monoclonal antibodies have been employed in investigative research to isolate, identify, and characterize molecular and cellular components of complex biological systems. In addition, monoclonal antibodies are increasing being utilized as highly specific reagents in the diagnosis of disease and are being tested for their effectiveness as biological response modifiers. Furthermore, monoclonal antibodies have been incorporated into numerous strategies to identify biological and/or chemical substances in tissue or environmental samples. The ability of antibodies to catalyze chemical reactions has recently been demonstrated suggesting that antibodies capable of performing enzymatic functions could be designed.

The development of monoclonal antibody technology has lead, during the last 20 years, to the generation of large panels of highly specific reagents that have had a tremendous impact on both the scope and pace of investigative and applied research. Indeed, monoclonal antibody technology has proven to be a important contributing factor in the growth of the biotechnology industry. It is likely that this trend will continue.

Introduction:

It has been known for over 100 years from the studies of von Behring and Kitazato that when a foreign substance is injected into a host, the host generates serum neutralizing substances (1). Neutralizing substances appear after a short lag period and are specific, in that they neutralize the injected substance but not an unrelated one. Another feature of interest in this host response is that it displays memory. Injection of the same substance a second time produces a response that has a shorter lag time and reaches a higher level. It was quickly determined that the repeated injection of a substance would result in a serum with high titers of neutralizing activity.

We now know that these neutralizing substances are antibody molecules and, in the serum of vertebrates, there are millions of distinct antibody molecules present. Antibody molecules are serum proteins with the same basic four chain (two heavy and two light chains) structure linked together by disulfide bonds. When the amino acid sequence of different antibodies were determined, it was found that there were regions of extensive homology (constant regions) and regions of dissimilarity (variable regions). The constant regions are localized toward the COOH terminal region of the molecule, while the variable regions are located at the NH2 terminus. The variable region is the critical site for antigen binding and is the distinctive feature that is unique to each individual antibody molecule.

An important idea that help understand how one can produce so many different antibodies was the clonal selection theory but forth by Burnett (2). In this theory, each antibody producing cell expresses but one antibody receptor, but within an individual, millions of different antibody forming cells exist. When an antigen engages those lymphocytes that have a receptor that can bind the antigen, those lymphocytes are activated, divide and differentiate into a plasma cell. Plasma cells are specialized cells that produce high levels of serum antibody. As a result, with time, there is an increase in the level of antibodies in the serum capable of binding to the antigen.

One important issue that need to be addressed was how an individual generates the millions of different antibodies capable of binding the universe of foreign antigens. The work of Susumu Tonegawa provided us with the key insight by determining that antibody genes are assembled in a unique fashion from minigene segments (3). Heavy chains are assembled from three minigenes termed V (variable), D (diversity) and J (joining) genes. There exist 100s of V-genes, ~20 D genes and ~6 J genes all of which can recombine to form a functional heavy chain gene. Since these genes randomly recombine, one can generate over 10,000 unique combinations. Light chain are similar but use only V and J-genes. Through the use of this unique genetic strategy, a given individual can generate millions of distinct antibody receptors. As a result one can begin to conceive of how the immune system is capable of generating antibody molecules capable of binding virtually any type of substance including proteins, nucleic acids, carbohydrate, lipid, or synthetic compounds.

Monoclonal antibodies: what are they and why are they useful?

Until the advent of monoclonal antibodies, investigators utilized the repeated injection of highly purified antigen to generate high-titered antibody reagents. Such reagents proved invaluable but had shortcomings since, the specific evoked antibody was only a fraction of the total antibody present. These "other" antibodies were either "natural antibodies" or antibodies evoked from other antigen stimulations. In addition, among the pool of specific antibodies, there was considerable heterogeneity in that antibodies with different affinities and/or reactivity to distinct structural domains on the molecule were common. Frequently this complexity produced unpredictable reactivities requiring the inclusion of large panels of controls to obtain meaningful information. The challenge was to purify a homogenous antibody preparation with a defined and specific reactivity. In some cases this was successful but it was soon realized that the ideal scenerio would be to "capture" an antibody producing cell in a manner that it is immortalized for tissue culture growth. In this manner, a single antibody molecule, with a defined specificity, could be identified, characterized and purified in large amounts.

Kohler and Millstein drew upon the filed of somatic cell genetics to devise hybridoma technology (4). In this approach, a myeloma cell rendered drug sensitive through mutation in a growth essential gene (HGPRT) is chemically fused with immune cells from a host immunized with the antigen of interest and the resulting cells are grown in medium containing the selective drug. Since the immune cells normally have a short life span in tissue culture and the myeloma cells are drug sensitive, the only cell that will survive are those myeloma cells which obtained a normal HGPRT gene from the immune cells. Such cells also have a high likelihood of also carrying the immune cell's antibody genes resulting in the generation of a "hybridoma" that can grow continuously in vitro and secrete a single monoclonal antibody. By using an appropriate screening strategy one can identify clones of cells secreting a single antibody of interest for subsequent expansion and isolation.

The development of monoclonal antibody technology offered a number of advantages over the original art of polyclonal antibodies. First, the generation of monoclonals is now a standard and increasingly routine procedure. Secondly, the use of impure antigen is tolerated since your ability to detect a monoclonal antibody of interest is largely dictated by your selection strategy. Thirdly, antibodies with selected properties (biological effects) or reactivities for specific structures could be selected. Lastly, since the hybridoma cells line is immortal, there is an unlimited source of the monoclonal antibody.

Monoclonal Antibodies: Applications:

Since Kohler and Millstein provided us with this approach some 20 years ago, monoclonal antibodies have impacted virtually every area of investigative science and applied research.

Perhaps the best example of the application of monoclonal antibodies is the generation of a large panel of reagents that define cell surface structures on the surface of bone marrow derived cells that give rise to and make up our protective immune system. Monoclonal antibodies that define CD molecules and flow cytometry have been used to identify the normal components of the immune system and to determine if these component are under-represented (in the case of immunodeficiency diseases) or over produced (in some cancers) (5). It is well known that a subset of lymphocyte termed T helper cells are important for normal immune function. Monoclonal antibodies reactive with the CD4 molecule expressed on helper cells was used to demonstrate that a decrease in CD4 cells is a feature of AIDS and the levels can be used to stage the disease. Thus, monoclonal antibodies have proven critical not only in allowing for an understanding of the complexity of the immune system but also in the understanding, diagnosis and management of disease.

Monoclonal antibodies have also been extensively used in the design of sensitive detection assays such as ELISAs. These tests have been used to detect normal and autoantibody levels, determine the presence and levels of autoantigens, viral/bacterial and other environmental antigens as well as assess the levels of normal components in bodily fluids (6).

Another area that monoclonals have had an impact on is the isolation and purification of molecules. A given monoclonal antibody can be coupled it to a insoluble surface and used to affinity purify the molecule of interest. In fact, this approach allows one to accomplish a several fold purification in a single step (7).

The field of molecular genomics has benefitted from the availability of monoclonal antibody technology. For example, a known monoclonal antibody that recognizes a molecule of interest can be used to identify its gene. Alternatively, if one has a newly identified gene with an unknown function, monoclonal antibodies can be generated against the predicted protein that it would encode and these reagents can be used for expression and function studies. These avenues also open up new windows of investigative research. For example, one can use monoclonal antibodies to determine if the gene is abnormally expressed in certain disease states or has a different structure in different individuals.

Monoclonal antibodies have been included in many protocols as biological response modifiers. Antibodies against bioactive cytokines have been used in therapy for many immunologically based diseases processes such as the control of transplantation rejection and the modulate of autoimmune diseases (8). There is an active interest in modifying monoclonal antibodies in a specific manner to be used as immunotoxins. Immunotoxins can be used to search out and destroy a specific cell type such as cells expressing a specific tumor marker that the antibody recognizes. This approach is currently being used in a number of different centers to attempt to attack certain tumors as well as being used by some to manipulate an immune response (9).

Because one has isolated a monoclonal antibody one can easily clone the immunoglobulin genes that encode it. This is a great advantage in that this would allow one to genetically tailor the monoclonal antibody for a particular use. For example, many monoclonal antibodies are generated in rodent species. The use of such reagents in humans is limited since the immune system can recognize the rodent antibody as foreign and mount a potent response against it. As a result of this issue there is considerable interest to "humanize" these antibodies by replacing those rodent structures with human counterpart and perhaps allow the reagent to be ignored by a human immune system.

Monoclonal antibodies have also been used in applied chemistry (10). It was appreciated that enzymes function in part by having a high affinity interaction with a short-lived transition state. It was reasoned that, if enzymes can interact with these transitional states, maybe an antibody can as well and potentially serve as an enzyme. By generating antibody reagents against enzyme inhibitors that mimic the transitional state one can isolate antibodies that can provide catalytic function. The catalytic reactions range from redox reactions to structural rearrangements. This initial success, together with the principles emerging in the field of combinatorial chemistry indicate that recognition molecules generated by the immune system have tremendous potential to be used as chemical tools.

In summary, it is safe to say that in 20 years monoclonal antibodies have had a tremendous impact on the biological and chemical sciences. Their availability has allowed investigators to ask new questions and to develop new insights and applications that will ultimately benefit human health and the human condition. While new technologies are being developed that may ultimately change how one proceeds to generate monoclonal reagents, it is clear that monoclonal antibodies will continue to have an important and positive impact on scientific endeavors in the near future.

References:

  1. Silverstein, A. M. (1989)A History of Immunology, Academic Press Inc. New York.
  2. Burnet, F. M. (1957) A modification of Jerne's theory on antibody production using the concept of clonal selection. Austral. J. Sci., 20: 67 - 69.
  3. Tonegawa, S. (1983) Somatic generation of antibody diversity. Nature 302: 575-581.
  4. Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495-497.
  5. Winkelstein, A. and Donnenberg, A. (1997) Clinical Application of Flow Cytometry. In Human Immunology (Eds. Leffell, M. S., Donnenberg, A. D., and Rose, N. R). CRC Press, New York.
  6. Rose, N., DeMacrio, E., Fahey, J., Friedman, H., Penn, G. (1997) Manual of Clinical Laboratory Immunology. American Soc. Microbiology Press, Washington, D.C.
  7. Coligan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M. and Strober, W. Eds. (1997) Current Protocols in Immunology. John Wiley & Sons Inc. Baltimore.
  8. Moller, G. (Ed) Antibodies in disease therapy. Immunol. Rev. 129: 1-201.
  9. Vitetta, E., Thorpe, P. E., Uhr, J. W. (1993) Immunotoxins: magic bullets or misguided missiles. Immunol. Today 14: 252-259.
  10. Schultz, P. G., and Lerner, R. A. (1995) From Molecular Diversity to Catalysis: Lessons from the Immune System. Science 269: 1835-1842.

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