Friday, September 20, 2013

A new ligand trap strategy to treat achondroplasia

Introduction

A very interesting study describing a new therapeutic strategy for achondroplasia has been published on Sep 18th. The study by the French group leaded by Dr. Elvire Gouze (1) represents a new approach and we will be briefly reviewing it here. 

Nevertheless, in order to let us understand how this new potential therapy works, I think it will worth to take a short ride through the mechanism causing achondroplasia. There are other articles in this blog reviewing the subject, but I always try to introduce new details or information, so we keep increasing our knowledge.

A bit about the molecular mechanism of achondroplasia

Achondroplasia is caused by a single switch (mutation) in the FGFR3 gene sequence, which generates the protein we all know as fibroblast growth factor receptor 3 (FGFR3). 

FGFR3 is what we call a receptor tyrosine kinase (an enzyme). It is called receptor because it is placed across the cell membrane of the chondrocyte to "wait" for the appropriate stimulus coming from outside the cell (Figure 1). In other words, as a receptor, FGFR3 "receives" signals from the body, like an antenna, and conducts these signals to the interior of the cell. 

Receptors are quite specific and will only respond to certain transmitters. In the case of FGFR3, only some molecules called fibroblast growth factors (FGFs) will be able to turn on the antenna. Indeed, previous works have found that FGFR3 has some "preferential" clients (or, as researchers call, ligands). From a family of 23 FGFs, it seems that FGF9 (2) and FGF18 (3) are the main ligands activating the FGFR3 antenna.

 Figure 1. FGFR3 is located across the cell membrane ready to receive signals from FGFs.




FGFR3 activation starts when two FGFs bind to two receptors. With the aid of a nearby molecule similar to heparin, called HSPG (from Heparan Sulphate Proteoglycan), the complex formed by the two FGFR3s and the two FGFs, called a dimer (Figure 2), will produce a rearrangement of the interior section of the two receptors, leading to the transfer of some phosphorus ions (I am simplifying), with the subsequent transfer of energy. This is the beginning of what we call signaling cascade or pathway (Figure 3). One after another, other neighbour proteins will be attracted and further direct the chemical signal to others till the signal is driven to the cell nucleus.(4)

Figure 2. The FGF-FGFR dimmer (from the Dr Moosa Mohammadi Lab website).

http://www.med.nyu.edu/mohammadi/LabPage/mv2.html

In normal conditions, the dimer will not last long. There are janitor systems detecting activated molecules to drive them for recycling or disintegration. They work as control systems to keep the cell chemical reactions in balance.

Figure 3. FGFR3 signaling cascade.



FGFR3 and achondroplasia

FGFRs are key proteins during the development of the new baby in utero. After birth however, although FGFR3 keeps being produced by several kinds of cells in the body, it looks like that the main role of this receptor is in the regulation of bone growth. Basically, FGFR3 is produced by chondrocytes located in very special regions found in the extremities of the long bones and in the other bones of the body, too, which are called growth plates

In normal conditions, acting together with several other proteins and agents, FGFR3 controls how chondrocytes proliferate, mature and give space for the new forming bone. In this complex concert FGFR3 is a natural brake, reducing the ability of chondrocytes to multiply and get old (mature).

However, in achondroplasia, the hallmark FGFR3 mutation G380R makes the dimer more stable, active for longer periods. This is enough to "overload" the cell nucleus with signals "saying": stop, do not multiply. This excessive signaling is the cause of the bone growth arrest we see in achondroplasia.

Stopping the workaholic receptor 

Since the blog started we have reviewed a lot of potential strategies to beat the super active FGFR3. You can find more information about them following the links provided on the top of this page, according to your preferential language. So, a large number of potential treatments are on the table, just waiting for investment. The study by Garcia et al. is just the newest published one. Let's review the key points of this study.

Trapping fibroblast growth factors (FGFs)

The concept brought by Dr Gouse and her group is that if we make a free form of FGFR3 available for binding with its ligands then less of these ligands would be binding the fixed FGFR3 (the FGFR3 located across the membrane) to generate the FGFR3 signaling cascade. On the contrary, since the free FGFR3 is not connected to the other intracellular proteins that respond to its activation, the signal is not transmitted at all. If the signal is not transmitted then FGFR3's negative effects on growth plate chondrocytes cease and the cells can multiply freely, restoring the growth pace.

We talked about a similar strategy some months ago in this article, in which the investigators created a molecule that is part of the FGFR1 and part an antibody, named GSK3052230 (formerly FP-1039), and explored it in several cancer models where FGFRs are important agents. This compound was able to prevent cancer cells to profit on the activation of FGFRs. (5) Importantly, the studies with this compound found that the ligands showing more susceptibility were exactly FGF9 and FGF18.

In the study by Garcia and colleagues, they worked with a soluble copy of the human FGFR3 (sFGFR3), which they injected twice a week in young growing animals bearing the same G380R mutation in FGFR3, thus having similar achondroplastic patterns such as short long bones, rounded cranium, spinal stenosis, etc.). 

They observed striking responses in all parameters measured. The treatment duration was three weeks, and mice exposed to sFGFR3 seemed to have apparent full recovery of growth (at least in the largest dose tested) (Figure 4). 

Figure 4. Treated and untreated FGFR3 G380R+ mice.

They examined the growth plates of untreated and treated control (wild type) and affected animals. They found that in the treated affected animals there was a marked recovery of the normal architecture of the growth plate. Even in the control animals, there was additional growth compared to the untreated control animals.

They also searched for evidence of sFGFR3 reaching the growth plates (remember that growth plates are thought to be a hard barrier for large molecules) and were able to detect sFGFR3 inside the growth plate.

They also verified if the expected ligands (FGF9 and FGF18) would bind to sFGFR3 and confirmed these ligands were trapped by the experimental compound. Furthermore, they also confirmed the kind of effect exerted by sFGFR3 showing that the classical FGFR3 cascade thought to be the responsible for its effects in chondrocytes was inhibited when the cells tested were exposed to sFGFR3 and those preferential ligands.

Finally, the researchers also examined several organs and tissues of the tested animals and found no signs of toxicity.

Questions 


The results are compelling, presenting a compound that seems to be at the same time efficient and safe. It is reasonable to say that the researchers were able to make a proof of concept that their compound does work. However, we couldn't help to notice some aspects. 

They tested two different doses in their animals (0.25mg/kg and 2.5mg/kg). When we check the animal pictures provided in the full paper version we can see that the animal treated with the lowest dose didn't achieve the same size of the control animal. By the other side, the affected animal treated with the largest dose grew more than the untreated control (Figure 2 in the full article). Moreover, the latter animal presented a more translucent spine than the treated control animal, in a fashion that reminded me the pattern seen in the animals tested with the continuous CNP exposure in the works by Yasoda and colleagues. (6) 

To respect the copyright of the images - I only post images here that are already available in the web, always citing the source - I can't put the picture of the Japanese mouse here, but I invite you to open the reference 6 and look at the picture provided in the article. Then compare both treated animals (you can compare with the animal B in the figure 4 above). It is possible that, depending on the dose used, sFGFR3 therapy could lead to overgrowth in affected animals, in the same way that continuous CNP loading seems to do, at least in mice.

Although the researchers have showed some evidence that sFGFR3 works by trapping FGF9 and FGF18, this should be further investigated. Since the interaction between FGFs, FGFR3 and HSPG request that they should be all in the same area it is important to understand the exact mechanism by how sFGFR3 is working. When the first tests with GSK3052230/FP-1039 were divulged, Dr Moosa Mohammadi, an expert in the field of FGF-FGFR molecular interactions, mentioned that the ligand trapping could not be the reason for the evidence of action of that compound, but that it would be competing against the natural receptor for the HSPGs.(7) The same could be happening with sFGFR3. Nonetheless, this doubt doesn't take the merit of this compound.

And, thinking out-of-the-box, this study also gives more evidence that it looks like that previous studies concluding that the growth plate would be a massive barrier for larger molecules might have missed some aspects of the biology of this structure. We reviewed this topic in this previous article, but there have been more evidence recently published showing that larger molecules can reach the chondrocytes inside the growth plate (8; reviewed here). Good opportunity to start reviewing these concepts.

Conclusion

I think the researchers presented a compelling potential therapeutic option to treat achondroplasia. There is still the need to learn which is the right dose to be further tested, how it will affect larger animals and how the body deals with it (how sFGFR3 is metabolised and eliminated) along with performing adequate safety tests before thinking in testing in humans. Some of these tests need the interest of biotechs or pharma industries to be accomplished. In this aspect, the other compound using the ligand trap concept, GSK3052230/FP-1039, has an advantage: it is already in phase 1. (9)

You might enjoy reading the article published by Science Now, authored by Mitch Leslie, which is the source of the figure 4 of this article and which provides a good coverage of the research with sFGFR3, including the opinion of key investigators in the field, such as Dr. William Horton.


Finally, a diligent parent of a child with achondroplasia (author of the Beyond Achondroplasia blog) found another project dealing with a distinct form of soluble FGFR3, this one being developed in US, funded by the NIH. You can find more information here.(10) No results have been published yet but the investigator describes that tests already performed also restored bone growth in the animal model.

References 

1. Garcia S et al. Postnatal soluble FGFR3 therapy rescues achondroplasia symptoms and restores bone growth in mice. Sci Transl Med 2013;5:203ra124.

2. Garofalo S et al.Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice. J Bone Miner Res 1999; 14 (11): 1909-15. Free access. 

3. Davidson D et al. Fibroblast growth factor (FGF) 18 signals through FGF Receptor 3 to promote chondrogenesis. J Biol Chem 2005; 280: 20509-15. Free access. 

4. Horton W. Molecular pathogenesis of achondroplasia. GGH 2006; 22 (4): 49-54. Free access.

5. Harding TC et al. Blockade of Nonhormonal Fibroblast Growth Factors by FP-1039 Inhibits Growth of Multiple Types of Cancer. Sci Transl Med 2013;5:178ra39. 

6. Yasoda A and Nakao K. Translational research of C-type natriuretic peptide (CNP) into skeletal dysplasias. Endocrine J 2010; 57 (8): 659- 66. Free access.

7. News and analysis. Deal watch: HGS and FivePrime in FGF 'ligand trap' deal. Nat Rev Drug Discov 2011;10(5):328. 

8. Ono K et al. The Ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation. Hum Mol Genet 2013; 22(15): 3048–62. doi: 10.1093/hmg/ddt162. 

9. Tolcher A et al. Preliminary results of a dose escalation study of the fibroblast growth factor(FGF) “trap” FP-1039 (FGFR1:Fc) in patients with advanced malignancies. 22nd EORTC-NCI-ACR symposium on molecular targets and cancer therapeutics, November 16-19, 2010. Berlin, Germany. Free access.

10. Ghivizzani SC. Delivery of soluble FGFR3 as a treatment for achondroplasia. National Institute of Arthritis and Musculoskeletal and Skin Diseases. 2013; Project Number: 5R01AR057422-04.

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