Antibodies and genetics

SIGNIFICANCE: Antibodies provide the main line of defense (immunity) in all vertebrates against infections caused by bacteria, fungi, viruses, or other foreign agents. Antibodies are used as therapeutic agents to prevent specific diseases and to identify the presence of antigens in a wide range of diagnostic procedures. Large quantities of antibodies have also been produced in plants for use in human and plant immunotherapy. Because of their importance to human and animal health, antibodies are widely studied by geneticists seeking improved methods of antibody production.

Antibody Structure

Antibodies are made up of a class of proteins called immunoglobulins (Ig’s) produced by plasma cells (descendants of activated B cells) in response to a specific foreign molecule known as an antigen. Most antigens are also proteins or proteins combined with sugars. Antibodies recognize, bind to, and inactivate antigens that have been introduced into an organism by various pathogens such as bacteria, fungi, and viruses.

The simplest form of molecule is a Y-shaped structure with two identical, long polypeptides (substances made up of many amino acids joined by chemical bonds) referred to as “heavy chains” and two identical, short polypeptides referred to as “light chains.” These chains are held together by chemical bonds. The lower portion of each chain has a constant region made up of similar amino acids in all antibody molecules, even among different species. The remaining upper portion of each chain, known as the “variable region,” differs in its sequence from other antibodies. The three-dimensional shape of the tips of the variable region (antigen-binding site) allows for the recognition and binding of target molecules (antigens). The high-affinity binding between antibody and antigen results from a combination of hydrophobic, ionic, and van der Waals forces. Antigen-binding sites have specific points of attachment on the antigen that are referred as “epitopes” or “antigenic determinants.”

Antibody Diversity

There are five classes of antibodies (IgG, IgM, IgD, IgA, and IgE), each having a distinct structure, size, and function. IgG is the principal immunoglobulin and constitutes about 80 percent of all antibodies in the serum.

The human body can manufacture a limitless number of antibodies, each of which can bind to a different antigen; however, human genomes have a limited number of genes that code for antibodies. It has been proposed that random of DNA segments is responsible for antibody variability. For example, one class of genes (encoding light chain) contains three regions: the L-V (leader-variable) region (in which each variable region is separated by a leader sequence), the J (joining) region, and the C (constant) region. In the embryonic B cells, each gene consists of from one hundred to three hundred L-V regions, approximately six J regions, and one C region. These segments are widely separated on the chromosome. As the mature, one of the L-V regions is randomly joined to one of the J regions and the adjacent C region by a recombination event. The remaining segments are cut from the chromosome and subsequently destroyed, resulting in a fusion gene encoding a specific light chain of an antibody. In mature B cells, this gene is then transcribed and translated into polypeptides that form a light chain of an antibody molecule. Genes for the other class of light chains as well as heavy chains are also made up of regions that undergo recombination during B-cell maturation. These random recombination events in each B cell during maturation lead to the production of billions of different antibody molecules. Each B cell has, however, been genetically programmed to produce only one of the many possible variants of the same antibody.

Production of Antibodies: Immune Response

Immunity is a state of bodily resistance brought about by the production of antibodies against an invasion by an antigen. The immune response is mediated by white blood cells known as that are made in the bone marrow. There are two types of lymphocytes: T cells, which are formed when lymphocytes migrate to the thymus gland, circulate in the blood, and become associated with lymph nodes and the spleen; and B cells, which are formed in bone marrow and move directly to the circulatory and the lymph systems. B cells are genetically programmed to produce antibodies. Each B cell synthesizes and secretes only one type of antibody, which has the ability to recognize with high affinity a discrete region (epitope or antigenic determinant) of an antigen. Generally, an antigen has several different epitopes, and each B cell produces a set of different antibodies corresponding to one of the many epitopes of the same antigen. All of the antibodies in this set, referred to as “polyclonal” antibodies, react with the same antigen.

The immune system is more effective at controlling infections than the nonspecific defense response (bodily defenses against infection—such as skin, fever, inflammation, phagocytes, natural killer cells, and some other antimicrobial substances—that are not part of the immune system proper). The immune system has three characteristic responses to antigens: diverse, which effectively neutralizes or destroys various foreign invaders, whether they are microbes, chemicals, dust, or pollen; specific, which effectively differentiates between harmful and harmless antigens; and anamnestic, which has a memory component that remembers and responds faster to a subsequent encounter with an antigen. The primary immune response involves the first combat with antigens, while the secondary immune response includes the memory component of a first assault. As a result, humans typically get some diseases (such as chicken pox) only once; other infections (such as cold and influenza) often recur because the causative viruses mutate, thus presenting a different antigenic face to the immune system each season.

An antibody-mediated immune response involves several stages: detection of antigens, activation of helper T cells, and antibody production by B cells. White blood cells known as macrophages continuously wander through the circulatory system and the interstitial spaces between cells searching for antigen molecules. Once an antigen is encountered, the invading molecule is engulfed and ingested by a macrophage. Helper T cells become activated by coming in contact with the antigen on the macrophage. In turn, an activated helper T cell identifies and activates a B cell. The activated T cells release cytokines (a class of biochemical signal molecules) that prompt the activated B cell to divide. Immediately, the activated B cell generates two types of daughter cells: antibody-producing cells (each of which synthesizes and releases millions of antibody molecules into the bloodstream in a single day) and (which have a life span of a few months to a year, depending on the immunoglobulin cell from which they derive). The B memory cells are the component of the immune memory system that, in response to a second exposure to the same type of antigen, produces antibodies in larger quantities and at faster rates over a longer time frame than the primary immune response. A similar cascade of events occurs when a macrophage presents an antigen directly to a B cell.

Polyclonal and Monoclonal Antibodies

Plasma cells originating from different B cells manufacture distinct antibody molecules because each B cell was presented with a specific portion of the same antigen by a helper T cell or macrophage. Thus a set of polyclonal antibodies is released in response to an invasion by a foreign agent. Each member of this group of polyclonal antibodies will launch the assault against the foreign agent by recognizing different epitopes of the same antigen. The polyclonal nature of antibodies has been well recognized in the medical field.

In the case of multiple myeloma (a type of cancer), one B cell out of billions in the body proliferates in an uncontrolled manner. Eventually, this event compromises the total population of B cells of the body. The immune system will produce huge amounts of IgG originating from the same B cell, which recognizes only one specific epitope of an antigen; therefore, this person’s immune system produces a set of antibodies referred to as “monoclonal” antibodies. Monoclonal antibodies form a population of identical antibodies that all recognize and are specific for one epitope on an antigen. Thus, someone with this condition may suffer frequent bacterial infections because of a lack of antibody diversity. Indeed, a bacterium whose antigens do not match the antibodies manufactured by the overabundant monoclonal B cells has a selective advantage.

The high-affinity binding capacity of antibodies with antigens has been employed in both therapeutic and diagnostic procedures. A manufacturing challenge remains, however: the effectiveness of commercial preparations of polyclonal antibodies can vary widely from batch to batch. In some instances of immunization, certain epitopes of a particular antigen are strong stimulators of antibody-producing cells, whereas at other times, the immune system responds more vigorously to different epitopes of the same antigen. Thus, one batch of polyclonal antibodies may have a low level of antibody molecules directed against a major epitope and not be as effective as the previous batch. To address such inconsistency between batches, researchers such as S. K. Rasmussen et al. have been developing methods in which desired monoclonal antibodies are simultaneously produced by multiple stable cell lines and then combined in a single-batch preparation.

It may instead be desirable to produce a that will produce with a high affinity for a specific epitope on the antigen for commercial use. Such a cell line would provide a consistent and continual supply of identical (monoclonal) antibodies. Monoclonal antibodies can be produced by hybridoma cells, which are generated by the fusion of cancerous B cells and normal spleen cells obtained from mice immunized with a specific antigen. After initial selection of hybridomas, monoclonal antibody production is maintained in culture. In addition, the hybridoma cells can be injected into mice to induce tumors that, in turn, will release large quantities of fluid containing the antibody. This fluid containing monoclonal antibodies can be collected periodically and may be used immediately or stored for future use. Various systems used to produce monoclonal antibodies include cultured lymphoid cell lines, yeast cells, Trichoderma reese (ascomycetes), insect cells, Escherichia coli, and monkey and Chinese hamster ovary cells. Transgenic organisms and plant cell cultures have been explored as potential systems for antibody expression.

Impact and Applications

The high-affinity binding capacity of antibodies may be used to inactivate antigens (within a living organism). The binding property of antibodies may also be employed in many therapeutic and diagnostic applications. In addition, it is a very effective tool in both immunological isolation and detection methods.

In 2020, researchers used mRNA technology to instruct cells to create a harmless protein spike to mimic those found on the surface of the SARS-CoV-2 virus, the virus responsible for the worldwide COVID-19 pandemic. The vaccine tricked the body’s immune system into creating antibodies that would fight the virus. In 2023, the National Institutes of Health used the same technology to develop a vaccine used to treat pancreatic cancer.

Monoclonal antibodies may outnumber all other products being explored by various biotechnology-oriented companies for the treatment and prevention of disease. For example, many strategies for the treatment of cancerous tumors as well as for the inhibition of human immunodeficiency virus (HIV) are based on the use of monoclonal antibodies. HIV is a retrovirus (a virus whose genetic material is ribonucleic acid, or RNA) that causes acquired immunodeficiency syndrome (AIDS). Advances in plant have made it possible to use transgenic plants to produce monoclonal antibodies on a large scale for therapeutic or diagnostic use. Indeed, one of the most promising applications of plant-produced antibodies in immunotherapy is in passive immunization (for example, against Streptococcus mutans, the most common cause of tooth decay). Large doses of the antibody are required in multiple applications for passive immunotherapy to be effective. Transgenic antibody-producing plants may be one source that can supply huge quantities of antibodies in a safe and cost-effective manner. It has been demonstrated that a hybrid IgA-IgG molecule produced by transgenic plants prevented colonization of S. mutans in culture, which appears to be how the antibody prevents colonization of this bacterium in vivo.

It has been estimated that antibodies expressed in soybeans at a level of 1 percent of total protein may cost approximately one hundred dollars per kilogram of antibody, which is relatively inexpensive in comparison with the cost of traditional antibiotics. Transgenic plants have also been used as bioreactors for the large-scale production of antibodies with no extensive purification schemes. In fact, antibodies have been expressed in transgenic tobacco roots and then accumulated in tobacco seeds. If this technology could be employed to obtain stable accumulation of antibodies in more edible plant organs such as potato tubers, it could potentially allow for long-term storage as well as a safe and easy delivery of specific antibodies for immunotherapeutic applications. In addition, plant-produced antibodies may be more desirable for human use than microbial-produced antibodies, because plant-produced antibodies undergo eukaryotic rather than the prokaryotic (bacterial) post-translational modifications. Human glycosylation (a biochemical process whereby sugars are attached onto the protein) is more closely related to that of plants than that of bacteria.

The potential use of antibody expression in plants for altering existing biochemical pathways has also been demonstrated. For example, germination mediated by a phytochrome (a biochemical produced by plants) has been altered by utilizing plant-produced antibodies. In addition, antibodies expressed in plants have been successfully used to immunize host plants against pathogenic infection; for example, tobacco plants have already been immunized with antibodies against viral attack. This approach has great potential to replace the traditional methods (use of chemicals) in controlling pathogens.

Key Terms

• B cellsa class of white blood cells (lymphocytes) derived from bone marrow responsible for antibody-directed immunity
• B memory cellsdescendants of activated B cells that are long-lived and that synthesize large amounts of antibodies in response to a subsequent exposure to the antigen, thus playing an important role in secondary immunity
• helper T cellsa class of white blood cells (lymphocytes) derived from bone marrow that prompts the production of antibodies by B cells in the presence of an antigen
• lymphocytestypes of white blood cells (including B cells and T cells) that provide immunity
• plasma cellsdescendants of activated B cells that synthesize and secrete a single antibody type in large quantities and also play an important role in primary immunity

Bibliography

"An mRNA Vaccine to Treat Pancreatic Cancer." National Institutes of Health, 23 May 2023, www.nih.gov/news-events/nih-research-matters/mrna-vaccine-treat-pancreatic-cancer. Accessed 9 Sept. 2024. "Antibody." National Human Genome Research Institute, 9 Sept. 2024, www.genome.gov/genetics-glossary/Antibody. Accessed 9 Sept. 2024.

Coico, Richard, and Geoffrey Sunshine. “Antibody Structure and Function.” Immunology: A Short Course. 6th ed. Hoboken: Wiley-Blackwell, 2009. 41–60. Print.

Dübel, Stefan, and Janice M. Reichert, eds. Handbook of Therapeutic Antibodies. 3 vols. Weinheim: Wiley-VCH, 2014. Print.

Glick, Bernard R., and Jack J. Pasternak, eds. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 4th ed. Washington: ASM, 2010. Print.

Harlow, Ed, and David Lane, eds. Using Antibodies: A Laboratory Manual. Rev. ed. Cold Spring Harbor: Cold Spring Harbor Laboratory P, 1999. Print.

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

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

Rasmussen, S. K., et al. "Recombinant Antibody Mixtures: Optimization of Cell Line Generation and Single-Batch Manufacturing Processes." BMC Proceedings, vol. 5, no. 8, 2011, doi: 10.1186/1753-6561-5-S8-O2. Accessed 9 Sept. 2024.

Raz, E. Immunostimulatory DNA Sequences. New York: Springer, 2001. Print.

Smith, Mathew D. “Antibody Production in Plants.” Biotechnology Advances 14.3 (1996): 267–81. Print.

Story, Lachel. "Body Defenses." Pathophysiology: A Practical Approach 2nd ed. Burlington: Jones, 2015. 31–50. Print.

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