Rituximab: The Promising Chemotherapy Agent As A Treatment For ME/CFS – A New And Large Norwegian Clinical Trial Begins
You would have by now heard of the buzz that has been surrounding the news of the 2009 and 2011 publications by Norwegian oncologists Dr Øystein Fluge and Professor Olav Mella in which they reported (in the latter trial publication) that two-thirds (10 out of 15) of ME/CFS patients receiving the chemotherapy treatment Rituximab reported significant improvement.
Not only did these results offer hope to ME/CFS patients worldwide they also gave an insight into the nature of the condition itself with the authors concluding that:
“The delayed responses starting from 2-7 months after Rituximab treatment, in spite of rapid B-cell depletion, suggests that CFS is an autoimmune disease and may be consistent with the gradual elimination of autoantibodies preceding clinical responses. The present findings will impact future research efforts in CFS.”
The new expanded randomised double blind trial began in September last year and is listed here at clinicaltrials.gov. One of the criticisms of the smaller 2011 trial was that it was not truly blinded. Rituximab as administered intravenously is opaque and the placebo in the 2011 trial was straight saline solution (which of course is a clear, colourless solution), therefore one could tell the placebo from Rituximab. However, this time the placebo has added (serum) albumin so the placebo and Rituximab will look identical.
Reports On The Norwegian Research And A Smaller UK Rituximab Trial
Cort Johnson reports in Simmaron Research on the new trial with responses to questions from Fluge and Mella and is an excellent description of the sub-parts to the trial.
At Science Based Medicine (Exploring issues & controversies in science & medicine) an article titled “Rituximab for Chronic Fatigue Syndrome: Jumping the Gun” takes a more critical view of the original Norwegian case series, small trial and new clinical trial. What the author describes is a doctor in the US who is treating ME/CFS patients with the expensive (it costs approx $US6000 per infusion) Rituximab before any of the clinical trials have been conducted, and there is evidence Rituximab really is the treatment for ME/CFS these patients hope it is. The author sounds a warning that ME/CFS sufferers should be cautious until there is conclusive proof Rituximab is shown to improve the condition – basically – it’s early days yet.
So What Is Rituximab?
Rituximab (traded as Rituxan, MabThera and Zytux) is a chemotherapy drug used to treat several types of cancer, including: non-Hodgkin’s lymphoma and chronic lymphocytic leukaemia (both types of blood cancer). Additionally when used in conjunction with methotrexate it is used to treat moderately to severely active rheumatoid arthritis.
As well as rheumatoid arthritis, Rituximab is widely used off-label to treat difficult cases of other autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus, Chronic inflammatory demyelinating polyneuropathy and autoimmune anemias.
Unlike some other small molecule chemotherapy drugs like cis-platin (PtCl2(NH3)2 – a single ion of platinum bonded to two chloride ions and two ammonia molecules, which is used to treat several cancers but most particularly testicular cancer), Rituximab is a very large (by comparison) protein molecule (See Figure 1).
Proteins are linear chains of amino acids that range from anywhere of a dozen or so amino acids (also referred to as peptides) – these smaller proteins are usually called polypeptides – to many thousands of amino acids. There are 21 common amino acids found in nature, they are all L-stereoisomers (“left handed” isomers). They are of the formula, H2NCHRCOOH, where R = the side chain by which each amino acid differs. The sequence of amino acids in the protein is referred to as its primary structure.
The side chains vary by charge, some are negatively/positively charged, some are neutral. Some are hydrophobic, some are hydrophilic. Some can participate in Hydrogen bonding, some cannot. The properties of the side chains determine how the proteins fold and determine their larger structure (secondary, tertiary and quaternary structures – as well as other factors) and how they interact with their surrounds and in the cases of enzymes and antibodies (vide infra), how they react/bond with their substrates and antigen epitopes respectively.
Figure 1. A view of the single-crystal X-ray crystal structure of Rituximab, showing the β-Sheets (yellow), α-Helices (pink) and other random structural sequences in grey. β-Sheets and α-Helices are the two main types of protein secondary structure [1,2]. The structure shows an epitope peptide (vide infra from CD20) bound in the Fab (antigen binding) region of the antibody – the small protein with two α-Helices at the top of the antibody.
Rituximab Is An Antibody
Rituximab, biochemically speaking, is what’s called a chimeric monoclonal antibody (where the -mab at the end of Rituximab comes from) against a trans-membrane protein called CD20 on the surface of B-lymphocytes (B-cells – a type of immune cell). By attaching selectively to these CD20 proteins on B-Cells, Rituximab can kill these cells. This is most desirable in cancers where there are too many B-cells, they are overactive or they are dysfunctional in some way.
Let me make an attempt at explaining some of that. Monoclonal antibodies are antibodies that bind to the same antigen (in our case here the CD20 protein), and are produced by identical B-cells (clones). In this case these cells come from mice.
So What Is An Antibody, And How Does It Work?
Let me start by saying the human immune system is extremely complex, if it wasn’t we’d all be dead from some pathogen or other. Also, if it wasn’t so complex, there would have been a cure for ME/CFS long ago. There’s still a lot not fully understood. An even brief summary of the immune system is beyond the scope of this post. I would direct interested readers to textbooks on the subject, such as a relatively “beginners” text “Understanding Immunology” by Peter Wood. For example, Monash University Library has both the 2nd and 3rd editions.
What is an antibody? Antibodies are (blood) serum soluble proteins that the B-cells (once they have differentiated into plasma cells) of our immune system (as opposed to T-cells and Natural Killer (NK) cells etc.) [3,4] produce to bind to antigens (they can be viruses, bacterial cell walls, an individual protein or other macromolecule) of foreign invaders and allow cells and other mechanisms of the immune system to recognise and eliminate them from our systems. Some antibodies remain bound to B-cells. There are also antigen receptors on the surfaces of T-cells.
Each antibody is specific to each antigen. Genetic variations in genes coding for the antibodies (vide infra) produce different combinations of antibodies, and effective antibodies (those that bind with greater affinity to an antigen and are hence good at doing their job at eliminating the threat) are selected to be synthesised in greater numbers and will proliferate.
What is amazing about this is the immune system stores this “information” in each type of antibody so if the body encounters the same antigen again sometime in the future, the secondary immune system “remembers” and activates production of the correct antibodies (assuming all is well of course). In this way we say the human body has developed an immunity to that particular antigen.
Antibodies, also known as immunoglobulins, themselves are generally large “Y” shaped glycoproteins (they have bound carbohydrates – sugars) of molecular weight around 150,000. There are five structural types (classes) of antibodies in humans based on this Y-shaped motif, these are IgA, IgD, IgE, IgG, and IgM, where Ig stands for Immunoglobulin. There are also further subclasses of some of these antibodies.
Let’s begin by describing a generic Y-shaped antibody (of the type IgD, IgG and IgE) (See Figure 2), consists of four polypeptide (protein) chains (two heavy chains and two light chains) connected by disulphide bonds. These chains have several domains. Figure 3 also shows a crystal structure of a real antibody IgG.
Figure 2. Basic schematic of an immunoglobulin/antibody molecule IgG. The two “arms” (the amino/NH3+ terminii of the peptides) of the Y-shape are where the antigens are bound in what are called the Variable Domains in the Fab regions (Fragment antigen binding). The Variable Domains are the most important regions for antigen binding – they bind in a lock-and-key mechanism with their appropriate antigen (more specifically the epitope – a particular molecular structure – on the antigen). The Fc (Fragment constant or historically also known as crystallisable) are at the carboxylate/COO- terminii of the peptides. Disulphide bonds are shown in yellow.
Figure 3. Single-crystal X-ray crystal structure of an IgG antibody showing several immunoglobulin domains making up the two heavy chains (red and blue) and the two light chains (green and yellow). The immunoglobulin domains are composed of between 7 (constant domains) and 9 (variable domains) β-sheets.
The two main regions of the monomeric Y-shaped IgG antibody are the Fc (Fragment constant – region) at the “base” of the Y and the Fab (Fragment antigen binding – region) at the “arms” of the Y. The Fc remains constant from subclass of IgG to IgG (which by the way is one of the most abundant proteins in the blood) and is not related to binding of the antigen. The two identical Fab regions at the “arms” of the Y are involved in the binding to the antigen in a lock-in-key mechanism. The lock-in-key mechanism is often seen in protein-protein or protein-substrate interactions, most significantly in enzyme-reactant interactions. It is the reason for an enzyme’s and in this case antibody’s specificity.
The “lock-in-key” mechanism is not as rigid as it sounds. Unlike that shown in Figure 3 where the antibody is shown in a single conformation in a crystal structure, in solution antibodies, proteins, molecules and other species are in a state of dynamic flux so that when they come together they undergo conformational changes so that the lock-in-key fit is really one of induced fit.
The side chains of the amino acids of the protein sequence in the VL (Variable Domains) at the antigen binding site are very specifically complementary in bonding (hydrogen bonding, electrostatic interactions and van der Waals interactions – types of non-covalent bonding involved in organic chemistry) capacity for their opposite number – the epitope (the protein sequence of the antigen to which they bind).
The reason antibodies can be generated with binding specificity for just one antigen is these Variable Domains (actually the sub-picture is a little more complex – there are hypervariable domains/complementarity determining regions (CDRs) – but I won’t go into them here). Genes coding for these Variable Domains change and code for new ones and so on and new specificities are born, without having to code for the whole molecule. Additionally, further variability is produced by different combinations of different heavy and light chains.
Not to be outdone, the Fc region also plays a part in the immune system. These are recognised by Fc receptors on other cells of the immune cells (such as neutrophils) so that when an antibody binds to an antigen, it is itself bound to one of these cells by the Fc receptor and the antibody and its uninvited guest is “eaten” by the cell and digested by enzymes within the cell (in a process called phagocytosis) and hence eliminated. There are other additional pathways the antigen-antibody complexes are eliminated.
Back To Rituximab
Rituximab (Figure 1) binds to the CD20 protein which is expressed on the surface (it spans the cellular membrane) of B-Cells (diseased and otherwise) and in a number of pathways that have been determined including attracting Natural Killer Cells to attack the B-Cell; and inducing apoptosis (the cell death pathway), results in depletion of B-cells, allowing a new population of healthy B-cells to develop from lymphoid stem cells.
Such a serious medication as Rituximab naturally has some serious (sometimes fatal) side effects , so it is by no means benign. Please see the following links for complete drug information:
David L Nelson; Albert L Lehninger; Michael M Cox, “Principles Of Biochemistry“, Fifth Edition, New York : W.H. Freeman, 2008.
Peter Wood, “Understanding Immunology“, Second Edition, Pearson Prentice Hall, 2006.
David Male, Jonathan Brostoff, David Roth and Ivan Roitt, “Immunology“, Seventh Edition, Mosby Elsevier, 2006.
Bradley, A. S., et al. “Altered functional B cell subset populations in patients with chronic fatigue syndrome compared to healthy controls.” Clin. Exp. Immunol., 2013, 172(1): 73-80.
Fluge, O., et al. “Benefit from B-lymphocyte depletion using the anti-CD20 antibody Rituximab in chronic fatigue syndrome. A double-blind and placebo-controlled study.” PLoS One, 2011, 6(10): e26358.
Fluge, O. and O. Mella. “Clinical impact of B-cell depletion with the anti-CD20 antibody rituximab in chronic fatigue syndrome: a preliminary case series.” BMC Neurol., 2009, 9.
Hauser, S. L., et al. “B-Cell Depletion with Rituximab in Relapsing–Remitting Multiple Sclerosis.” New England Journal of Medicine, 2008, 358(7): 676-688.
Smith, M. R. “Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance.” Oncogene, 2003, 22(47): 7359-7368.