Friday, November 15, 2019

Report on rituximab as a biologic

Report on rituximab as a biologic Rituximab is a chimeric monoclonal antibody that has been one of the most prevalent biologics on the market, and has made some very significant sales over the last few years. It was originally designed in 1986 by IDEC pharmaceuticals. The drug is now being marketed under the brand names Rituxin and MabThera, dependant on the region. The antibody is chimeric in nature and is comprised of murine and human regions. It contains heavy and light variable regions of a murine origin and constant heavy and light regions of human origins meaning the Fc domain is of human origins, and so can invoke Fc receptor binding (see figure 1). The antibody is produced artificially and the gene construct of the murine and human domains are engineered and inserted into vectors, and then expressed in mammalian cells line. The mammalian cell line of choice for this specific drug is the Chinese Hamster Ovary cells (CHO cells); these provided the corrected folded drug in sufficient quantities. The use of the mammalian cell line ensured that firstly the antibodies had the correct codon usage, glycosylation, amino acids and folding, as these are mammalian cells so are able to carry out these tasks to closer specification as human cells. Figure 1: representation of the rituximab antibody. Taken from The filtrate from the suspension had to be purified to obtain the rituximab that will be used for clinical applications. There are two methods used to perform this task. First of all Ion-exchange chromatography, this method allows the removal to toxic products, cell mass, some viruses and unimportant protein products. The second step of purification that is used is affinity chromatography; affinity chromatography is then used to remove the specific antibody with high resolution and specificity. As a mammalian cell line was used for the expression there is a slight chance that possible viruses can pass through the purification process and be found in the final product, so a few processes were introduced to remove these possible contaminants. The media was autoclaved and human transferrin was removed, the ion exchange chromatography also helps remove some retroviruses. The drug reaches a purity of around 99%, and has a shelf stability of 2 years. The FDA originally approved Rituximab in 1997 for use against B-Cell non-Hodgkin lymphoma that had become resistant to all other forms of treatment (chemotherapy). Since its approval for use against lymphoma, it has been approved and is used off label for use against other diseases. It is seen to have an efficacy against autoimmune (AI) diseases, transplant rejection treatment and severe ulcerative colitis. Rituximab is known as an anti-CD20 antibody, meaning that it acts on and binds to the CD20 molecule. CD20 molecules are found in large amount on the surface of activated B-cells. The reason for targeting these cells is that the activated B-cells are generally the ones that are causing the problems in the disease discussed earlier. However, CD20 is also expressed, in smaller amounts, on most other B-cells so there is a certain amount of collateral damage to the immune system, which in some cases has led to an increase risk of catching serious infections. CD20 is a very weird antigen, it has no known natural ligand and its function is still very much unknown, opinions are that it helps maintain calcium concentrations across the membrane. After being binding to the CD20 at amino acid positions 170-13 and 183-183, it is seen to halt the cell cycle and induce apoptosis. There are two methods that carry out this apoptotic response, the first being the indirect killing of the cell. This involves a mechanism called anti-body dependant cell cytoxicity (ADCC). ADCC is mechanism where one the FAB part of the antibody has bound to the receptor on the surface the Fc domain changes formation and can now bind to an FC receptor. Fc receptors are found on other cells within the immune system, such as NK cells, the binding of the FC domain to one of these cells induces a killing response from the activated immune cell recruited. The Fc domain also has the ability to activate complement, so the biding of specific blood proteases, induces lysis of the cell. There is also a less well-understood mechanism that rituximab uses to kill activated B cells. This is by directly inducing apoptosis upon binding, why this occurs is not fully unde rstood, from studies it can be seen that the binding of rituximab to the CD20 causes a down regulation of MHC, a down regulation of the B-cell receptor and flux down pathways that induce apoptosis. In a recent study it can be seen that 50% increase of 30%. So that is a huge increase in the amount of patients who were currently unresponsive to available treatments. As can be seen from these results the introduction of rituximab was a real breakthrough in the field, and provided a method of treatment for those people who had no other option. As motioned earlier the drug is frequently used off label by clinicians. Rituximab has been seen to have very promising result when used to treat MS and some other AI diseases. There have also been recent developments into the treatment of severe ulcerative colitis, and rituximab is currently in phase III trials for use against ulcerative colitis where existing treatments are currently not effective. Rituximab was and is still a huge commercial success; grossing $5.68 billion in 2009 (a 9% increase on the previous year) it was one of the best selling biologics. With all the possible applications of this drug it does mean that there is still a commercial future for the drug. Rituximab is currently being marketed by Roche, Biogen and other companies have exclusivity for marketing in Japan, China and India This can be seen with how often clinicians use the drug off label to treat diseases that the FDA didnt originally approve it for. The shear wide-ranging applications of the drug are one of the main reasons for its commercial success. The drug was also a huge boost to the clinicians tools to fight against diseases which had become resistant or unresponsive to the existing gold standards, such as in B cell lymphoma and RA as discussed above. There is also still future prospects for this drug with phase III trials currently underway for the use of the drug against severe ulcerative c olitis and possible uses in replaced rejection treatment plans, so it seems that this drug is likely to be a block buster for some time to come.

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