Author Archives: Mirella Bucci

Our immune system is a killing machine. It consists of various types of specialized cells and proteins that function to destroy invaders and “non-self” or mutated “self” proteins, such as those that come from virus78_RH image_optes, bacteria, and cancer cells. In the autoimmune diseases such as the P/P diseases, this mechanism has gone awry and the immune system actually attacks its own cells.

In P/P patients, antibodies generated by B cells of the immune system block the function of desmoglein proteins Dsg1 and Dsg3 known to be important in binding together keratinocytes of the skin and mucous membranes, but it is not known how the rogue antibodies are generated by the immune system, how they escape the quality control mechanisms in place that only allow B cells with non-“self” specificities to survive, and why P/P patients are so rare.

New research led by Dr. Aimee Payne in the Department of Dermatology at the University of Pennsylvania (Nature Communications, http://www.nature.com/ncomms/2014/140619/ncomms5167/abs/ncomms5167.html) helps us begin to understand why.

In previous work, Dr. Payne and colleagues have identified antibodies that recognize Dsg1 and Dsg3 (so-called anti-Dsg1 and anti-Dsg3 antibodies) and have also identified regions of those antibodies that are important for the ability of those antibodies to be pathogenic — that is, to recognize their Dsg targets in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) and to disrupt their function. To extend this work and to better understand how PV autoantibodies arise, Dr. Payne and colleagues have performed a similar analysis of PV patients.

PV patients can present as either mucosal-dominant, where only the mucous membranes are affected or as mucocutaneous, affecting both the mucous membranes and the skin. Almost all mucosal-dominant PV patients have anti-Dsg3 autoantibodies, while the mucocutaneous patients have anti-Dsg3 autoantibodies as well as anti-Dsg1 autoantibodies. Since it is thought that Dsg1 and Dsg3 can compensate for each other’s function, the presence of functional Dsg1 in the skin in the presence of anti-Dsg3 autoantibodies can explain why mucosal-dominant patients do not have skin lesions.

The authors first isolated the full antibody repertoire from four different untreated PV patients, all with mucocutaneous disease. They isolated and characterized these in a multistep process that ultimately allowed them to determine the amino acid compositions (by cloning and DNA sequencing methods) of the patient’s PV antibodies. This led to the assignment of six unique antibodies from Patient 1 and five additional unique antibodies from the three other PV patients.

In total, the sequencing efforts identified 21 unique heavy chains among the four patients.

All 11 antibodies could bind to Dsg3 and this was mediated via a domain (called EC1) in Dsg3 that is known to be important for its adhesive interactions, suggesting that anti-Dsg3 autoantibody binding to Dsg3 leads to a direct block in Dsg3 function in keratinocytes (and subsequent skin blistering).

Curiously, not all of the antibodies that the authors identified that bound Dsg3 could cause blistering when added to human skin tissue samples; the VH1-46-containing antibodies did. They determined that these differences in functional effects were due to the inability of the nonpathogenic antibodies to bind to the functional domains of Dsg3.

Even more curiously, the authors found that all four patients had at least one PV antibody that consisted of an identical variable region termed VH1-46. They also found very little change in the VH1-46 amino acid sequence in the patient antibodies compared to the known sequence of VH1-46 that also exists in unaffected patients (considered the “wild-type” or germline sequence).

As noted by the authors, this is a pattern typical of a somatically mutated antibody sequence, meaning that very few changes were generated during the development of the B cells (each with its own single antibody that it makes, see Figure).

They did some additional experiments to define the ability of those amino acid changes to affect the binding to Dsg3. They conclude that VH1-46 autoantibodies in PV are generated during B cell development and require very little mutation to become pathogenic. This suggests that they appear early during the development of the disease and explains their prevalence in all of the patients tested here.

These autoantibodies may not be the most common later on (during full-blown disease), but they may provide a clue to why and how pemphigus arises. The ability of these autoantibodies to escape the quality control machinery at play during B cell development is likely due to the low levels of Dsg3 antigen available that would distinguish these antibodies as “self” antibodies and therefore the ability of the machinery to mark the cells and their rogue autoantibodies for destruction.

These data led the authors to speculate that the five pathogenic (disease-causing) VH1-46 anti-Dsg3 mAbs that they’ve identified in this study are among the earliest autoantibodies formed in PV patients, caused only by how simple they are to generate from germline sequences. They also define a mechanism for how these autoantibodies are made and most importantly, how they are missed by the quality control machinery – all low probability events that likely account for the rarity of PV.

664715_11160870-pillRare diseases, including several autoimmune disorders, are getting more attention from drug-makers, according to a new report by the Pharmaceutical Research and Manufacturers of America (PhRMA), a consortium of 36 US-based pharmaceutical and biotechnology companies. In 2012 alone, 13 drugs for orphan diseases (“orphan drugs”) were approved by the Food and Drug Administration (FDA). Approximately 452 medicines and vaccines are in development for the nearly 7,000 orphan diseases worldwide.

orphan diseases are defined as diseases with fewer than 200,000 patients. In total, however, across the nearly 7,000 orphan diseases, 30 million people in the US, or about 10% of the population, are affected by an orphan disease. The pemphigus and pemphigoid (P/P) diseases are considered “ultra-orphan” diseases because they are extremely rare. It is estimated there are only about 50,000 new P/P cases each year worldwide, with only a few thousand of those being in the US.

Rare diseases tend to be more complex than common diseases, meaning that there are a number of factors that combine to cause disease. In the case of P/P, while there seem to be genetic risk factors, how these contribute, singularly or in combination, and to what extent the environment (like diet and other conditions that are present) also contributes is not well understood.

Somewhat fortuitously, complex diseases represent the next great frontier for drug developers. Having tapped into the ‘simpler’ diseases, making great strides in treatment of conditions like high cholesterol, these ‘low-hanging fruit’, as drug-makers like to call them, have been consumed. It is truly a time of paradigm-shifting mentality among drug makers.

That said, the costs of developing new medicines is extremely high, so companies must make their choices wisely. If we were to calculate the amount that pharmaceutical and biotechnology companies spend on research and development yearly and compare that to the number of drugs that are approved for clinical use by the FDA each year, the cost per successful drug is a staggering $1.2 billion. It’s not difficult to imagine, then, why companies aimed at developing new drugs are most interested in those that can recoup these huge costs — for instance, by developing drugs for very common conditions and risk factors such as diabetes and high cholesterol. As well, given the complex nature of rare diseases, they are not necessarily among the ‘low-hanging fruit’ that some diseases represent.

To incentivize companies to prioritize new drugs for rare conditions, they may apply for orphan drug status through the FDA, a result of passage of the Orphan Drug Act (ODA) of 1983. With this status, a drug receives seven years of market exclusivity. Market exclusivity is particularly appealing to companies developing drugs because the seven-year exclusivity period differs from laws applicable to other drugs in that it does not begin until the drug is approved by the FDA approval.

The ODA is considered a resounding success. Since its inception, there have been more than 400 medicines approved for a total of 447 orphan diseases. As well, there are hundreds of new medicines in development, including an impressive list available in the PhRMA 2013 report (phrma.org/sites/default/files/pdf/Rare_Diseases_2013.pdf).

While not all of the 452 orphan drugs in development will be approved for patient use, this is certainly a lot of activity. A search of the list included within the PhRMA report, as well as a search of clinicaltrials.gov (that lists all clinical trials in progress), shows a handful of drugs in testing for conditions related to or directed at P/P.

There are 18 new orphan drugs in phase I-III trials (there are three phases of clinical trials and drugs must pass all of them, indicating reasonable levels of safety and meaningful efficacy-effectiveness in treating the condition) that are indicated for autoimmune disorders.

New drugs are not the only source of treatment for disease. Another source is to use an existing drug, developed for another condition, for a different indication. Such is the case with Rituxan® (rituximab), which was originally developed for Non-Hodgkin’s lymphoma. In that disease, B cells of the immune system bearing a marker called CD20 (thus the name CD20+ B cells) have gone awry.

Since P/P shares this hallmark, Rituxan® has been successfully used ‘off-label’ for P/P. It is an antibody-based drug, which requires it to be injected into the patient. In general, any drug that acts as a suppressor of the immune system (immunosuppressant) is a potential candidate for treating a range of auto-immune conditions, including P/P. CellCept® (mycophenolate mofetil), another immune system suppressor that was developed for transplant patients to help prevent the body’s rejection of the ‘foreign’ organ, has recently been approved for use in P/P.

Besides the high cost of developing new drugs, companies that seek treatments for orphan diseases face difficulty in finding enough patients to participate. Indeed, patients tend to be dispersed geographically and may include small children. Physicians and patients who are interested in participating in trials or gaining more information should visit clinicaltrials.org.

Within the P/P community, the IPPF is also a great resource for learning about clinical trials. Members of our medical advisory board serve as investigators on trials and being in our patient database could lead to a company reaching out to you about participating in a trial.

For instance, among the new drugs aimed at treating P/P, drug-maker Novartis is studying VAY736, an antibody-based drug aimed at another B cell marker called BAFF-R. The study is in a very early stage and should be recruiting patients soon.

The time is ripe for development of new drugs for complex orphan diseases. The surge in new medicines in the first 30 years since the ODA should accelerate as less ‘low-hanging fruit’ exist for companies developing new drugs.

According to a recent scientific article published by Dr. Sergei Grando (IPPF Medical Advisory Board Vice Chairperson) and some of his colleagues from the University of California, Irvine, “The ultimate goal of pemphigus research is to develop an effective treatment modality that would allow patients to achieve and maintain clinical remission without the need for systemic corticosteroids.” This represents the next great horizon in treating the disease since the use of corticosteroids was implemented in the 1950s. Prior to that, patients were not expected to live more than five years after the onset of their disease. So we’ve come a long way but goals like that of the Grando research group are lofty indeed.

To begin to develop new treatment strategies for any disease, it is important to better understand the underlying biology that causes the disease and that is associated with disease physiology. Targeting pathways with drugs is the ultimate goal and it is all the better if the drugs used are specific to these pathways as this will limit potential side-effects associated with their use. This would seem to exclude the use of steroids such as those that are a standard of treatment currently. In their current work, the groups of Dr. Grando and Dr. Ping Wang (Journal of Biological Chemistry, http://www.jbc.org/cgi/doi/10.1074/jbc.M113.472100) examine the effects of antibodies (IgGs) known to be present in PV patients and find that they deleteriously affect specific functions of the mitochondria of skin cells (keratinocytes). The mitochondria are the compartments within cells where all of the energy, in the form of ATP, is generated.

Protecting mitochondria, the authors believe, should help to alleviate the cell death that is associated with PV.

IgGs produced in PV cause keratinocytes to die due to their being “split” apart or detached from each other within the epithelial layers of the skin (fact check). However, the mechanisms by which IgGs cause this splitting and in fact, whether there are more than one type of IgG generated in PV has not been determined. Previous work from Dr. Grando’s group has contributed to a theory where various antibodies that bind to keratinocytes, including the well-described anti-desmoglein antibodies, work together to cause the cellular effects that lead to PV.

As well, previous work has implicated the mitochondria in PV. Indeed, the mitochondria that have been tested from lesions of PV patients are defective in many of their key functions. These include maintaining a balance of antioxidants and limiting the production of reactive oxygen species (ROS) that lead to untold cellular damage.

The current paper solidifies the model that multiple targets of keratinocytes (both on the surface – the desmogleins, and inside – the mitochondria) are at play in PV. As well, it suggests that multiple antibody types are involved in the end result – cell death. The antibodies that the authors focused on are called mitochondrial antibodies (MtAbs) because of their ability to enter keratinocytes and bind to mitochondrial proteins. MtAbs make up what may be the most important class of IgGs in PV patients. Removing MtAbs from the serum of PV patients makes the serum incapable of causing keratinocyte detachment. Serum is what remains of the blood after you remove all of the cells – including proteins, antibodies and small molecules from metabolism. The authors have now found that the IgGs from the serum of PV patients can cause the mitochondrial dysfunction seen in previous work.

These IgG mixtures, which contain the MtAbs, cause numerous changes in the vital functions of mitochondria. For instance, they saw an increase in the production of ROS from keratinocytes, a decline in ATP production, and changes in the mitochondrial membrane potential, a hallmark of the tidy cell death pathway called apoptosis. This is the first time scientists have shown such dramatic changes in mitochondrial functions with patient IgGs. Even more striking is that compounds that protect mitochondria could help the keratinocytes resist the adverse effects of the IgGs. These compounds, minocycline, nicotinamide (a well-known over-the-counter antioxidant supplement), and cyclosporine A have previously been used, often in combination, with beneficial effects on PV patients, but an understanding of why they are effective hasn’t been clear until now.

Since these three mitochondria-protecting drugs are already in use in some PV patients, the authors argue that optimizing their use, by determining at what levels they need to be dosed in individual patients, for starters, should make them an ideal non-steroid treatment for PV.