Hybridomas and monoclonal antibodies

SIGNIFICANCE: In 1975, Georges Köhler and Cesar Milstein reported that fusion of spleen cells from an immunized mouse with a cultured plasmacytoma cell line resulted in the formation of hybrid cells called hybridomas that secreted the antibody molecules that the spleen cells had been stimulated to produce. Clones of hybrid cells producing antibodies with a desired specificity are called monoclonal antibodies and can be used as a reliable and continuous source of that antibody. These well-defined and specific antibody reagents have a wide range of biological uses, including basic research, industrial applications, and medical diagnostics and therapeutics.

A New Way to Make Antibodies

Because of their specificity, antisera have long been used as biological reagents to detect or isolate molecules of interest. They have been useful for biological research, industrial separation applications, clinical assays, and immunotherapy. One disadvantage of conventional antisera is that they are heterogeneous collections of antibodies against a variety of antigenic determinants present on the antigen that has elicited the antibody response. In an animal from which antisera is collected, the mixture of antibodies changes with time, so that the types and relative amounts of particular antibodies are different in samples taken at different times. This variation makes standardization of reagents difficult and means that the amount of characterized and standardized antisera is limited to that available from a particular sample.

The publication of a report by Georges Köhler and Cesar Milstein in the journal Nature in 1975 describing production of the first monoclonal antibodies provided a method to produce continuous supplies of antibodies against specific antigenic determinants. Milstein’s laboratory had been conducting basic research on the synthesis of immunoglobulin chains in plasma cells, mature that produce large amounts of a single type of immunoglobulin. As a model system, they were using rat and mouse plasma cell tumors (plasmacytomas). Prior to 1975, Köhler and Milstein had completed a series of experiments in which they had fused rat and mouse plasmacytomas and determined that the light and heavy chains from the two species associate randomly to form the various possible combinations. In these experiments they used mutant plasmacytoma lines that would not grow in selective culture media, while the hybrid cells complemented each others’ deficiencies and multiplied in culture.

After immunizing mice with sheep red blood cells (SRBC), Köhler and Milstein removed the spleen cells from the immunized mice and fused them with a mouse plasmacytoma cell line. Again, the selective media did not allow unfused plasmacytomas to grow, and unfused spleen cells lasted for only a short time in culture so that only hybrids between plasmacytoma cells and spleen cells grew as hybrids. These hybrid plasmacytomas have come to be called hybridomas.

Shortly after the two types of cells are fused by incubation with a fusing agent such as polyethylene glycol, they are plated out into a series of hundreds of small wells so that only a limited number of hybrids grow out together in the same well. Depending on the frequency of hybrids and the number of wells used, it is possible to distribute the cells so that each hybrid cell grows up in a separate well.

On the basis of the number of spleen cells that would normally be making antibodies against SRBC after mice have been immunized with them, the investigators expected that one well in about 100,000 or more might have a clone of hybrid cells making antibody that reacted against this antigen. The supernatants (liquid overlying settled material) from hundreds of wells were tested, and the large majority were found to react with the immunizing antigen. Further work with other antigens confirmed that a significant fraction of hybrid cells formed with spleen cells of immunized mice produce antibodies reacting with the antigen recently injected into the mouse. The production of homogeneous antibodies from clones of hybrid cells thus became a practical way to obtain reliable supplies of well-defined immunological reagents.

The antibodies can be collected from the media in which the cells are grown, or the hybridomas can be injected into mice so that larger concentrations of monoclonal antibodies can be collected from fluid that collects in the abdominal cavity of the animals.

Specific Antibodies Against Antigen Mixtures

One advantage of separating an animal’s antibody response into individual antibody components by and separation of cells derived from each fusion event is that antibodies that react with individual antigenic components can be isolated even when the mouse is immunized with a complex mixture of antigens. For example, human tumor cells injected into a mouse stimulate the production of many different types of antibodies. A few of these antibodies may react specifically with tumor cells or specific types of human cells, but, in a conventional antisera, these antibodies would be mixed with other antibodies that react with any human cell and would not be easily separated from them. If the tumor cells are injected and hybridomas are made and screened to detect antibodies that react with tumor cells and not with most normal cells, it is possible to isolate antibodies that are useful for detection and characterization of specific types of tumor cells. Similar procedures can also be used to make antibodies against a single protein after the mouse has been immunized with this protein included in a complex mixture of other biological molecules such as a cell extract.

Following the first report of monoclonal antibodies, biologists began to realize the implications of being able to produce a continuous supply of antibodies with selected and well-defined reactivity patterns. There was discussion of “magic bullets” that would react specifically with and carry specific cytotoxic agents to tumor cells without adverse effects on normal cells. Biologists working in various experimental systems realized how specific and reliable sources of antibody reagent might contribute to their investigations, and entrepreneurs started several companies to develop and apply monoclonal antibody methods. This initial enthusiasm was quickly moderated as some of the technical difficulties involved in production and use of these antibodies became apparent; with time, however, many of the projected advantages of these reagents have become a reality.

Monoclonal Reagents

A survey of catalogs of companies selling products used in biological research confirms that many of the conventional antisera commonly used as research reagents have been replaced with monoclonal antibodies. These products are advantageous to the suppliers, being produced in constant supply with standardized protocols from hybrid cells, and the users, who receive well-characterized reagents with known specificities free of other antibodies that could produce extraneous and unexpected reactions when used in some assay conditions. Antibodies are available against a wide range of biomolecules reflecting current trends in research; examples include antibodies against cytoskeletal proteins, protein kinases, and proteins, gene products involved in the transition of normal cells to cancer cells such as those involved in apoptosis.

Immunologists were among the first to take advantage of monoclonal antibody technology. They were able to use them to “trap” the spleen cells making antibodies against small, well-defined molecules called haptens and to then characterize the antibodies produced by the hybridomas. This enabled them to define classes of antibodies made against specific antigenic determinants and to derive information about the structure of the antibody-binding sites and how they are related to the determinants they bind. Other investigators produced antibodies that reacted specifically against subsets of lymphocytes playing specific roles in the immune responses of animals and humans. These reagents were then used to study the roles that these subsets of immune cells play in responses to various types of antigens.

Antibodies that react with specific types of immune cells have also been used to modulate the immune response. For example, antibodies that react with lymphocytes that would normally react with a transplanted tissue or organ can be used to deplete these cells from the circulation and thus reduce their response against the transplanted tissue.

Monoclonal Antibodies as Diagnostic Reagents

Monoclonal antibodies have been used as both in vitro and diagnostic reagents. By the 1980s, many clinical diagnostic tests such as assays for hormone or drug levels relied upon antisera as detecting reagents. Antibodies reacting with specific types of bacteria and viruses have also been used to classify infections so that the most effective treatment can be determined. In the case of production of antibodies for typing microorganisms, it has frequently been easier to make type-specific monoclonal antibodies than it had been to produce antisera that could be used to identify the same microorganisms.

Companies supplying these diagnostic reagents have gradually switched over to the use of monoclonal antibody products, thus facilitating the standardization of the reactions and the protocols used for the clinical tests. The reproducibility of the assays and the reagents has made it possible to introduce some of these tests that depend upon measurement of concentrations of substances in urine as kits that can be used by consumers in their own homes. Kits have been made available for testing glucose levels of diabetics, for pregnancy, and for the presence of certain drugs.

Although the much-hoped-for “magic bullet” that would eradicate cancer has not been found, there are several antibodies in use for tumor detection and for experimental forms of cancer therapy. Monoclonal antibodies that react selectively with cancer cells but not normal cells can be used to deliver cytotoxic molecules to the cancer cells. Monoclonal reagents are also used to deliver isotopes that can be used to detect the presence of small concentrations of cancer cells that would not normally be found until the tumors grew to a larger size.

Since 1986, when the Food and Drug Administration (FDA) approved the first therapeutic monoclonal antibody for allograft rejection in renal transplants, more than thirty other monoclonal antibodies have been approved, and hundreds more are undergoing clinical trials. Most of these are used in the treatment of cancers or autoimmune diseases such as Crohn disease or rheumatoid arthritis. During this time, monoclonals have been particularly effective in the treatment of Hodgkin’s lymphoma and other lymphoid malignancies.

Human Monoclonal Antibodies

Initially, the majority of monoclonal antibodies made against human antigens were mouse antibodies derived from the spleens of immunized mice. When administered to humans in clinical settings, the disadvantage of the animal origin of the antibodies soon became apparent. The human immune system recognized the mouse antibodies as foreign proteins and produced an immune response against them, limiting their usefulness. In addition, the mouse antibodies were unable to carry out certain immune functions such as effectively binding to human Fc receptors. Even when the initial response to an antibody’s administration was positive, the immune reaction against the foreign protein quickly limited its effectiveness. In an attempt to avoid this problem, human monoclonal antibodies have been developed using several methods. The first is the hybridization of human lymphocytes stimulated to produce antibodies against the antigen of interest with mouse plasmacytomas or later with human plasmacytoma cell lines. This method has been used successfully, although it is limited by the ability to obtain human B cells or plasma cells stimulated against specific antigens, because it is not possible to give an individual a series of immunizations and then remove stimulated cells from the spleen. Limited success has resulted from the fusion of circulating lymphocytes from immunized individuals or fusion of lymphocytes that have been stimulated by the antigen in cell cultures. Investigators have reported some success in making antitumor monoclonal antibodies by fusing lymph node cells from cancer patients with plasmacytoma cell lines and screening for antibodies that react with the tumor cells.

There has also been some success at “humanizing” mouse antibodies using molecular genetic techniques. In this process, the portion of the genes that make the variable regions of the mouse antibody protein that reacts with a particular antigen is spliced in to replace the variable region of a human antibody molecule being produced by a cultured human cell or human hybridoma. What is produced is a human antibody protein that has the binding specificity of the original mouse monoclonal antibody. When such antibodies are used for human therapy, the reaction against the injected protein is reduced compared to the administration of the whole mouse antibody molecules. A variation on this method is the production of chimeric antibodies by exchanging the variable domain from a mouse antibody with the desired specificity with the human variable domain from a human antibody of the desired Ig class.

Another application of antibody engineering is the production of bispecific antibodies. This has been accomplished by fusing two hybridomas making antibodies against two different antigens. The result is an antibody that contains two types of binding sites and thus binds and cross-links two antigens, bringing them into close proximity to each other.

Recombinant Antibodies

Advances in molecular genetic techniques and in the characterization of the genes for the variable and constant regions of antibody molecules have made it possible to produce new forms of monoclonal antibodies. The generation of these recombinant antibodies is not dependent upon the immunizing of animals but on the utilization of combinations of antibody genes generated using the in vitro techniques of genetic engineering. Geneticists discovered that genes inserted into the genes for fibers expressed on the surface of bacterial viruses called bacteriophages are expressed and detectable as new protein sequences on the surface of the bacteriophage. Investigators working with antibody genes found that they could produce populations of bacteriophage expressing combinations of antibody-variable genes. Molecular genetic methods have made it possible to generate populations of bacteriophage expressing different combinations of antibody-variable genes with frequencies approaching the number present in an individual mouse or human immune system. The population of bacteriophage can be screened for binding to an antigen of interest, and the bacteriophage expressing combinations of variable regions binding to the antigen can be multiplied and then used to generate recombinant antibody molecules in culture.

As phage display technology was further developed and useful antibodies derived, it was found that random mutagenesis of the isolated antibody gene could also be used to derive a panel of mutant binding sites with higher affinity binding than the antibody detected in the original screening.

Recombinant DNA technology has also made it possible to modify the procedures for immunization and production of human monoclonal bodies. A process referred to as DNA immunization involves introducing the gene for for the target antigen in a form that results in the expression of the protein and an immune response against it. Also mice that have had their own immunoglobulin genes replaced by the corresponding human genes can be immunized to produce human monoclonal antibodies.

Researchers have also experimented with introducing antibody genes into plants, resulting in plants that produce quantities of the specific antibodies. Hybridomas or bacteriophages expressing specific antibodies of interest may be a potential source of the antibody gene sequences introduced into these plant antibody factories.

Monoclonal Antibodies in Proteomics

Coincident with the development of genomic methods for determination of at the RNA level has been an interest in detection of relative levels of protein expression. Incorporation of monoclonal antibodies into microarrays that allow the comparison of the expression of proteins from different cells or tissues has since been developed and will likely be important in both basic research and clinical assays as this technology continues to be developed.

Key Terms

• antibodya protein produced by plasma cells (matured B cells) that binds specifically to an antigen
• antigena foreign molecule or microorganism that stimulates an immune response in an animal
• antiseraa complex mixture of heterogeneous antibodies that react with various parts of an antigen; each type of antibody protein in the mixture is made by a different type (clone) of plasma cell
• plasmacytomaa plasma cell tumor that can be grown continuously in a culture

Bibliography

Chames, Patrick, et al. “Therapeutic Antibodies: Successes, Limitations, and Hopes for the Future.” British Journal of Pharmacology 157.2 (2009): 220–33. Print.

Dahan, Sophie, et al. “Antibody-Based Proteomics: From Bench to Bedside.” Proteomics Clinical Applications 1.9 (2007): 922–33. Print.

Gibbs, W. W. “Plantibodies: Human Antibodies Produced by Field Crops Enter Clinical Trials.” Scientific American 277.5 (1997): 44. Print.

Hoogenboom, H. R. “Designing and Optimizing Library Selection Strategies for Generating High-Affinity Antibodies.” Trends in Biotechnology 15.2 (1997): 62–70. Print.

Kontermann, Roland, and Stefan Dübel, eds. Antibody Engineering. 2nd ed. New York: Springer, 2010. Print.

Mayforth, Ruth D. Designing Antibodies. San Diego: Academic, 1993. Print.

Mohta, Alpana. "Understanding Hybridoma Technology for Monoclonal Antibody Production." The Scientist, 9 May 2023, www.the-scientist.com/autoimmune-diseases-an-alternative-application-for-immunotherapy-72108. Accessed 4 Sept. 2024.

Ossipow, Vincent, and Nicolas Fischer. Monoclonal Antibodies: Methods and Protocols. 2nd ed. New York: Humana, 2014. Print.

Springer, Timothy. Hybridoma Technology in the Biosciences and Medicine. New York: Springer, 2013. Print.

Stigbrand, T., et al. “Twenty Years with Monoclonal Antibodies: State of the Art.” Acta Oncologica 35.3 (1996): 259–65. Print.

Van de Winkel, J. G., et al. “Immunotherapeutic Potential of Bispecific Antibodies.” Immunology Today 18 (Dec. 1997): 562–74. Print.

Wang, Henry Y., and Tadayuki Imanaka, eds. Antibody Expression and Engineering. Washington, DC: Amer. Chemical Soc., 1995. Print.