Report on rituximab as a biologic

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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. http://www.nature.com/onc/journal/v22/n47/images/1206939f1.jpg

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Figure 1: representation of the rituximab antibody. Taken from http://www.nature.com/onc/journal/v22/n47/fig_tab/1206939f1.html

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 it's 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 understood, 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 <90% of the peripheral cells are killed, 60-70% of the lymphatic cells and around 1-2% of the b cells in the bone marrow, so with these results it is obviously necessary to repeat the infusion every 4/6 weeks to ensure that all the incorrectly activate B cells are killed/removed from the immune system. (Figure 2 shows the killing process diagrammatically)

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http://stke.sciencemag.org/content/sigtrans/vol2004/issue241/images/large/2412004pe30F1.jpeg

Figure 2: a Cartoon representation of the possible mechanism of killing induced by Rituximab. Taken from http://stke.sciencemag.org/content/sigtrans/vol2004/issue241/images/large/2412004pe30F1.jpeg

With all the boasts that rituximab had, there was still an obvious need to carry out tests to demonstrate firstly its efficacy as a drug, its purity and quality and of paramount importance its safety and tolerability.

During the safety trial 282 patients were involved in the study, and two cumulative dosage levels were assessed. From the patients studied <50% experienced adverse effects after the first infusion, the effects were caused from cytokine release or chemical mediator release syndrome. The meant that these patients were experiencing symptoms such as fever, chills, angioeanima and in approx. 10% of cases hypotension was also a symptom.

During the safety trials there were also infections seen, such as common viral and bacterial infections, which were not commonly seen in chemotherapy. The infections occurred in around 17% of patients, and were considered not to be serious or severe infections. In the combinational study, of rituximab plus CHOP chemotherapy, there was also no evidence of any adverse effects from using them in combination. There were also some fatalities during the trial the details of which are in table 1.

Table 1: lit of fatalities along with the causes.

The efficacy studies can be seen in the table below (table 2). They include the data for patients you exhibit a partial response (PR) and complete response (CR). From the data in the table you can see that there were good rates of response to the drug. In the second trail ( II) only the data for patients who had had some response were taken further, so that the true efficacy of the drug could be ascertained. So the drug produced CR in around 50-60% of patients, a huge increase in patients that were not currently responding to treatment.

Table 2: A table showing the efficacy rates of rituximab.

As the drug as been approved for a while and been around since 1997 there have been many clinical studies, all of which have produced similar results to the ones I have described below. The drug was first approved for use against b-cell non-Hodgkin lymphoma, so the phase III trials were first carried out on patients with this disease. The results given below are from a study by the EORC (referenced below). The trial was a randomised double blind trial. They found that when you used rituximab against its gold standard, CHOP chemotherapy, that the patients who had been administered with rituximab had a higher rate of remission of the disease. It was also discovered that a combinational treatment plan increased the induction of remission in patients from 72% to 85%, and statistical tests showed that the results were significant. The progression free survival was also increased significantly when rituximab was included in the treatment plan.

Rituximab was approved in 2006 by the FDA for use against Rheumatoid Arthritis (RA) that is still active after methotrexate treatment. The phase III clinical trails that were conducted included 161 patients and was a randomised double blind test. The drug was being tested against the gold standard for RA treatment methotrexate. The trial included using both treatments by themselves and then a combinational therapy in other patients. The trails measured the effectiveness of the drug in the trails was decided by how many of the patients had seen > 50% improvement in their symptoms. With rituximab alone there was and increase of 20% and in a combinational therapy there was an increase in the amount of patients seeing > 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.

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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 didn't 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 colitis 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.