Monoclonal antibodies (mAb or moAb) are mono-specific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitopes.
Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug name ends in –mab
Production of monoclonal antibodies involving human–mouse hybrid cells was described by Jerrold Schwaber in 1973. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies fuse them with healthy antibody-producing B-cells, and select the successfully fused cells. In 1988, Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies, removing the reactions that many monoclonal antibodies caused in some patients.
Hybridoma cell production (Hybridoma technology)
Monoclonal antibodies are typically made by fusing myeloma cells with the spleen cells
from a mouse that has been immunized with the desired antigen. However, recent advances have allowed the use of rabbit B-cells to form a Rabbit Hybridoma. Polyethylene glycol is used to fuse adjacent plasma membranes, but the success rate is low so a selective medium in which
only fused cells can grow is used. This is possible because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), an enzyme necessary for the salvage synthesis of nucleic acids. The absence of HGPRT is not a problem for
these cells unless the de novo purine synthesis pathway is also disrupted. By exposing cells to aminopterin (a folic acid analogue, which inhibits dihydrofolate reductase, DHFR), they are unable to use the de novo pathway and become fully auxotrophic for nucleic acids requiring
supplementation to survive.
The selective culture medium is called HAT medium because it contains Hypoxanthine,
Aminopterin, and Thymidine. This medium is selective for fused (Hybridoma) cells. Unfused myeloma cells cannot grow because they lack HGPRT, and thus cannot replicate their DNA. Unfused spleen cells cannot grow indefinitely because of their limited life span. Only fused hybrid cells, referred to as hybridomas, are able to grow indefinitely in the media because the
spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal (similar to a cancer cell).
This mixture of cells is then diluted and clones are grown from single parent cells on microtitre wells. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen (with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot. The most productive and stable clone is then selected for future use.
- The hybridomas can be grown indefinitely in a suitable cell culture medium.
- They can also be injected into mice (in the peritoneal cavity, surrounding the gut).
There, they produce tumors secreting an antibody-rich fluid called ascites fluid.
The medium must be enriched during in-vitro selection to further favour Hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone. Culture-medium conditioned by macrophages can also be used. Production in cell culture is usually preferred as the ascites technique is painful to the animal. Where alternate techniques exist, this method (ascites) is considered unethical.
Purification of monoclonal antibodies
After obtaining either a media sample of cultured hybridomas or a sample of ascites fluid, the desired antibodies must be extracted. The contaminants in the cell culture sample would consist primarily of media components such as growth factors, hormones, and transferrins. In contrast, the in vivo sample is likely to have host antibodies, proteases, nucleases, nucleic
acids, and viruses. In both cases, other secretions by the hybridomas such as cytokines may be present. There may also be bacterial contamination and, as a result, endotoxins that are secreted
by the bacteria. Depending on the complexity of the media required in cell culture, and thus the contaminants in question, one method (in vivo or in vitro) may be preferable to the other.
The sample is first conditioned, or prepared for purification. Cells, cell debris, lipids, and clotted material are first removed, typically by centrifugation followed by filtration with a 0.45 µm filter. These large particles can cause a phenomenon called membrane fouling in later purification steps. In addition, the concentration of product in the sample may not be sufficient, especially in cases where the desired antibody is one produced by a low-secreting cell line. The sample is therefore condensed by ultra-filtration or dialysis.
Most of the charged impurities are usually anions such as nucleic acids and endotoxins.
- These are often separated by ion exchange chromatography.
a. either cation exchange chromatography is used at a low enough pH that the desired
antibody binds to the column while anions flow through, or
b. anion exchange chromatography is used at a high enough pH that the desired antibody flows through the column while anions bind to it. Various proteins can also be separated out along with the anions based on their isoelectric point (pI). For example, albumin has a pI of 4.8, which is significantly lower than that of most monoclonal antibodies, which have a pI of 6.1. In other words, at a given pH, the average charge of albumin molecules is likely to be more negative. Transferrins, on the other hand, have pI of 5.9, so cannot easily be separated out by this method. A difference in pI of at least 1 is necessary for a good separation. - Transferrins can instead be removed by size exclusion chromatography. The advantage of this purification method is that it is one of the more reliable chromatography techniques. Since we are dealing with proteins, properties such as charge and affinity are not consistent and vary with pH as molecules are protonated and deprotonated, while size stays relatively constant. Nonetheless, it has drawbacks such as low resolution, low
capacity and low elution times. - A much quicker, single-step method of separation is Protein A/G affinity
chromatography. The antibody selectively binds to Protein A/G, so a high level of purity (generally >80%) is obtained. However, this method may be problematic for antibodies that are easily damaged, as harsh conditions are generally used. A low pH can break the bonds to remove the antibody from the column. In addition to possibly
affecting the product, low pH can cause Protein A/G itself to leak off the column and appear in the eluted sample. Gentle elution buffer systems that employ high salt concentrations are also available to avoid exposing sensitive antibodies to low pH. Cost is
also an important consideration with this method because immobilized Protein A/G is a more expensive resin. - To achieve maximum purity in a single step, affinity purification can be performed, using the antigen to provide exquisite specificity for the antibody. In this method, the
antigen used to generate the antibody is covalently attached to an agarose support. If the antigen is a peptide, it is commonly synthesized with a terminal cysteine, which allows selective attachment to a carrier protein, such as KLH during development and to the
support for purification. The antibody-containing media is then incubated with the immobilized antigen, either in batch or as the antibody is passed through a column, where it selectively binds and can be retained while impurities are washed away. An elution with a low pH buffer or a more gentle high salt elution buffer is then used to recover purified antibody from the support. - To further select for antibodies, the antibodies can be precipitated out using sodium sulfate or ammonium sulfate. Antibodies precipitate at low concentrations of the salt, while most other proteins precipitate at higher concentrations. The appropriate level of salt is added in order to achieve the best separation. Excess salt must then be removed by a desalting method such as dialysis.
The final purity can be analyzed using a chromatogram. Any impurities will produce peaks, and the volume under the peak indicates the amount of the impurity. Alternatively, gel electrophoresis and capillary electrophoresis can be carried out. Impurities will produce bands of varying intensity, depending on how much of the impurity is present.
Antibody heterogeneity
Product heterogeneity is common to monoclonal antibody and other recombinant biological production and is typically introduced either upstream during expression or downstream during
manufacturing.
These variants are typically aggregates, deamidation products, glycosylation variants, oxidized amino acid side chains, as well as amino and carboxyl terminal amino acid additions. These seemingly minute changes in a monoclonal antibody’s structure can have a profound effect on
preclinical stability and process optimization as well as therapeutic product potency, bioavailability, and immunogenicity. The generally accepted method of purification of monoclonal antibodies includes capture of the product target with Protein A, elution, acidification to inactivate potential Mammalian viruses, followed by cation exchange chromatography, and finally anion exchange chromatography.
Displacement chromatography has been used to identify and characterize these often unseen variants in quantities that are suitable for subsequent preclinical evaluation regimens such as animal pharmacokinetic studies. Knowledge gained during the preclinical development phase is
critical for enhanced understanding of product quality and provides a basis for risk management and increased regulatory flexibility. The recent Food and Drug Administration’s Quality by Design initiative attempts to provide guidance on development and to facilitate design of
products and processes that maximizes efficacy and safety profile while enhancing product manufacturability.
Recombinant Monoclonal Antibodies Production
The production of recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different
amino acid sequences from which antibodies with desired specificities can be selected. The phage antibody libraries are a variant of the phage antigen libraries first invented by George Pieczenik. These techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale.
Chimeric Antibodies
Definition: Antibodies that is composed of genetically different tissues, either naturally or as a result of a laboratory procedure.
Early on, a major problem for the therapeutic use of monoclonal antibodies in medicine was that initial methods used to produce them yielded mouse, not human antibodies. While structurally similar, differences between the two were sufficient to invoke an immune response when murine monoclonal antibodies were injected into humans, resulting in their rapid removal from the
blood, as well as systemic inflammatory effects, and the production of human anti-mouse antibodies (HAMA).
In an effort to overcome this obstacle, approaches using recombinant DNA have been explored since the late 1980s. In one approach, mouse DNA encoding the binding portion of a monoclonal antibody was merged with human antibody-producing DNA in living cells. The expression of
this chimeric DNA through cell culture yielded partially mouse, partially human monoclonal antibodies. For this product, the descriptive terms “chimeric” and “humanised” monoclonal antibody have been used to reflect the combination of mouse and human DNA sources used in
the recombinant process.
‘Fully’ Human Monoclonal Antibodies
Ever since the discovery that monoclonal antibodies could be generated, scientists have targeted the creation of ‘fully’ human antibodies to avoid some of the side effects of humanized or chimeric antibodies. Two successful approaches have been identified: transgenic mice and phage display.
Transgenic mice technology is by far the most successful approach to making ‘fully’ human monoclonal antibody therapeutics. Seven(7) of the 9 ‘fully’ human monoclonal antibody therapeutics on the market were derived in this manner.
Transgenic mice have been exploited by a number of commercial organisations:
- Medarex — who marketed their UltiMab platform. Medarex were acquired in July 2009 by Bristol Myers Squibb
- Abgenix — who marketed their Xenomouse technology. Abgenix were acquired in April 2006 by Amgen.
- Regeneron’s VelocImmune technology.
- Kymab – who market their Kymouse technology.
One of the most successful commercial organisations using phage display technology was Cambridge Antibody Technology (CAT). Scientists at CAT demonstrated that phage display could be used such that variable antibody domains could be expressed on filamentous phage
antibodies.
Applications of Monoclonal Antibodies
Monoclonal antibodies have been generated and approved to treat cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection.
Other applications of monoclonal antibodies include:
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Diagnostic tests:
Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence of this substance. The Western blot test and immuno dot blot tests detect the
protein on a membrane. They are also very useful in immunohistochemistry, which detect antigen in fixed tissue sections and immunofluorescence test, which detect the substance in a frozen tissue section or in live cells.
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Monoclonal antibody therapy
- Autoimmune diseases: Monoclonal antibodies used for autoimmune diseases include infliximab and adalimumab, which are effective in rheumatoid arthritis, Crohn’s disease
and ulcerative Colitis by their ability to bind to and inhibit TNF-α. Basiliximab and daclizumab inhibit IL-2 on activated T cells and thereby help prevent acute rejection of kidney transplants. Omalizumab inhibits human immunoglobulin E (IgE) and is useful in moderate-to-severe allergic asthma. - Pathogen neutralization and antiviral therapy. Antibody binding can directly and effectively block the activity of many pathogens, often without requiring Fc-mediated cytotoxicity. Indeed, this has always been the promise of antibody-mediated viral neutralization. The first monoclonal antibody for the treatment of viral disease, Synagis, was approved by the FDA in 1998 (Table 1). Synagis is a humanized antibody used for the prevention of severe respiratory syncytial virus (RSV) disease. Despite this success, and the wide range of antibodies available against human immunodeficiency type 1 (HIV) and herpes simplex virus (HSV), the use of recombinant antibodies as therapeutics for viral infection has been limited.
- Intracellular antibodies. Antibody fragments can be expressed as intracellular proteins, typically as scFvs termed intrabodies, and equipped with targeting signals either to neutralize intracellular gene products or to target cellular pathways. Intrabodies also have important antiviral potential, particularly through their targeting of intracellular action to mandatory viral proteins such as the Vif, Tat or Rev Components of HIV72. Antibody frameworks have been adapted that substantially improve expression levels and solubility in the intracellular reducing environment. The expression of intrabodies in vivo can be encoded into gene therapy vectors, and this could ultimately be their most powerful clinical application.
- Vaccine applications. Troy-bodies are engineered vaccine antibodies containing cryptic T-cell epitopes to enhance antigen presentation. Troy-bodies effectively target antigen presenting cells (APCs) and, after processing, expose cryptic T-cell epitopes to direct Tcell activation. In the preferred format, the Fv domain provides APC specificity and the C domains encode the cryptic T-cell epitopes. These new vaccines can be redesigned to target many different APCs and enhance immunity to many different T-cell epitopes. Alternative vaccine strategies include the use of engineered APC-targeted antibodies that direct adenoviruses to deliver vaccine-inducing epitopes as a gene therapy capsule and B7-targeted scaffolds (scFv and VL domains) that enable antigen-loading of dendritic cell.
- Cancer treatment: One possible treatment for cancer involves monoclonal antibodies that target only cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate. It is also possible to design bispecific monoclonal antibodies that can bind with their Fab regions to both target antigen (in this case, cancer cells) and to a conjugate or effector cell. Every intact antibody can bind to cell receptors or other proteins with its Fc region.
Monoclonal antibodies for cancer include:
- ADEPT, antibody directed enzyme prodrug therapy
- ADCC, antibody dependent cell-mediated cytotoxicity
- CDC, complement dependent cytotoxicity
- MAb, monoclonal antibody;
- scFv, single-chain Fv fragment.
References
- Monoclonal Antibodies Overview. Genescript. Accessed August 8, 2021
- Monoclonal Antibodies. Medicine Net. Accessed August 8, 2021
- Monoclonal Antibodies. Wikipedia. Accessed August 8, 2021
- Monoclonal Vs polyclonal Antibodies. Proteintech. Accessed August 8, 2021
- Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus. World Journal of Hepatology. Accessed August 8, 2021