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Introduction

Antibody issue

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"Learn from yesterday, live for today, hope for tomorrow. The important thing is not to stop questioning." - Albert Einstein

Antibody production

The current production methods of antibodies involve the use of laboratory animals, by exploiting their immune system's ability to generate antibodies. The current technique, the hybridoma technology, is the most widely used method for producing monoclonal antibodies (mAb). Antibodies are produced by B-lymphocytes of the adaptive immune system Adaptive ImmunityThe part of the immune system that is mediated by lymphocytes and activated by foreign molecules. The difference from this system and the innate system is the ability to develop memory and react at repeated exposure to the same sepcific microbe.. Antibodies are highly specific proteins that remain surface-bound on the B-lymphocyte until it recognizes a foreign molecule like bacterial or viral proteins, generally termed an antigen. An antibody will recognize and bind a specific region of the antigen, called an epitope. Reference: Lubert Stryer JMB, John L. Tymoczko. The Immune System. Biochemistry. 7 ed: W. H. Freeman and Company; 2012. The antibody will be changed from a surface-bound receptor molecule to a soluble and secreted molecule by an alternative splicing mechanism of the pre-mRNA transcript of the antibody gene.

Figure 1: The maturation of an B-lymphocyte in reaction to a specific microbe.

Activated B-lymphocytes only produce one type of antibody that has affinity for a single epitope on an antigen. The antibody structure consists of a constant part and a hypervariable part. The hypervariable part is responsible for the specificity towards the epitope of an antigen. The hypervariable part is generated by rearrangement of gene segments during development of B-lymphocytes and by RNA splicing. Reference: Lubert Stryer JMB, John L. Tymoczko. The Immune System. Biochemistry. 7 ed: W. H. Freeman and Company; 2012. Furthermore, protein processing contributes to diversity and affinity of the produced antibodies. A somatic mutation Somatic mutationWhen mutations occur in somatic cell lines the mutation will be present in all descendants of this particular cell within the organism. These mutations can lead to malignant cancer. can greatly increase affinity for the antigen, a process called affinity maturation. Reference: Lubert Stryer JMB, John L. Tymoczko. The Immune System. Biochemistry. 7 ed: W. H. Freeman and Company; 2012. Every B-lymphocyte processes differently, thus every B cell will produce different antibodies. This increases the antibody repertoire and ensures the best possible protection from intruders.

Applications of antibodies

Figure 2: Immunostaining of α-tubulin of the microtubuli network of Retina Pigment Epithelia (RPE) cells. DNA stained with DAPI (4',6-diamidino-2-phenylindole)

Antibodies are highly complex yet elegant and sophisticated molecules, designed to protect an organism against invaders. Its highly specific nature and ability to bind a target with high affinity can be exploited in many ways Reference: Cowan MK. Host Defenses II - Specific Immunity and Immunization. Microbiology - A Systems Approach. 3 ed: McGraw-Hill; 2012. e.g. as antivenoms AntivenomThe word venom is derived from Latin venenum, meaning poison. Venom is milked from the relevant source, e.g. snake or spider and then injected into a horse, sheep, rabbit or mouse. Immunization of the animal will result in production of antibodies against the antigen, which can be harvested from the blood. can greatly increase affinity for the antigen, a process called affinity maturation in treatment of snakebites. Reference: Chippaux JP, Goyffon M. Venoms, antivenoms and immunotherapy. Toxicon. 1998;36(6):823-46. Antibodies function in a preventive manner through passive immunization of patients by injection of ready antibodies, instead of inducing production, as in a normal vaccine. Reference: Clem AS. Fundamentals of vaccine immunology. J Glob Infect Dis. 2011;3(1):73-8. Additionally they can be used to detect presence of a specific protein (its antigen) – which makes them useful in diagnostics (so-named Immuno-diagnostics). Reference: Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-7. They are used in techniques such as Western Blotting, ELISA ELISAEnzyme-Linked ImmunoSorbent Assay and when conjugated to a fluorescent protein they can be used to localize proteins within cells (called immunohistochemistry, or specifically Immunolacalization), which is how images like the one in Figure 2 is obtained. Reference: Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-7.

The ability of antibodies to bind their target can be useful in treatment of cancer. Antibodies that bind and inhibit growth factors or block a hyperactive signalling pathway can potentially block tumor progression. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30.

Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev Cancer. 2012;12(4):278-87. It has also been proposed to use antibodies as a drug delivery system by conjugating (linking) the antibody to a chemotherapeutic drug. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30.

BioPharma P. What Are Humanized Monoclonal Antibodies 2015 [cited 2015 08/08] (Link) Currently only few antibody-based treatments are available. Cancer cells have antigens on their surface which can be cancer specific or present at a higher concentration than on normal cells. These can potentially be targeted by antibodies. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30.

The hope is that human-like antibodies against cancer antigens will be able to activate the immune system that in turn will eliminate the cancer. It is no surprise why scientists, researchers and doctors are interested in this sophisticated molecule. However, the available methods for producing antibodies are less sophisticated and elegant, ethically speaking, and stains the reputation of antibody production.

Industrial production of monoclonal antibodies using the hybridoma technology

Figure 3: The Hybridoma Technology.

The hybridoma technology, illustrated in Figure 3, was originally developed by Kohler and Milstein in 1975 and relies on an animals ability to generate antibodies against a foreign molecule. The animal of choice in production of monoclonal antibodies is typically a mouse or a rabbit. This production method involves 6 general steps;

Figure 4: Chimeric and humanized antibodies.

Immunization: Over several weeks the animal is continuously injected with antigen. Often the animals are also injected with an adjuvant that enhances the immune response. Reference: Stills HF, Jr. Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants. ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 2005;46(3):280-93.
Isolation: Euthanizing and extracting the blood (in polyclonal antibody production) and spleen from the animal and isolation of the antibody producing B-lymphocytes.
Fusion: Fusion of the B-lymphocytes from the spleen and a murine myeloma cell line (B-lymphocyte cancer cells), thus creating immortal and high-yield antibody producing hybrid cells.
Screening: Affinity based screening tests of the different antibodies to select the most effective one. This involves techniques such as ELISA and are followed by affinity chromatography purification. Reference: Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-7.
Multiplication: Growing of selected hybridoma cell in a culture dish or transplantation of the tumor cells into a new animal for a higher yield. The latter is the typical procedure.
Harvesting: The hybridoma cells will mass produce their antibody and mass divide. This causes tumor development and accumulation of fluids in the abdominal cavities (ascites). The fluid can be drained and the antibodies harvested from it.

Antibodies generated in the murine model will have to be further modified before they can be used therapeutically in humans. The murine antibodies will be recognized as foreign in humans and will elicit an immune response against murine antibody. Before mouse antibodies (or any other animal antibody) can be used in clinical medicine, they have to be 'humanized'. Humanized animal antibodies are also known as chimeric antibodies limited Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30. and are generated by combining the constant part of a human antibody with the variable (epitope-recognizing) part of the murine antibody. However chimeric antibodies are still about 30% foreign for the human body, and therefore they elicit significant immunogenicity in humans. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30. Creation of chimeric and humanized antibodies is not as easy as it might sound. It requires extensive knowledge and characterisation of the antibody structure and effective methods are still limited. Reference: Berger M, Shankar V, Vafai A. Therapeutic applications of monoclonal antibodies. Am J Med Sci. 2002;324(1):14-30.

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Antibody production

Applications of antibodies

Industrial production of monoclonal antibodies using the hybridoma technology