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# # # # Antibodies are tiny y-shaped markers used by the immune system to label foreign agents within the body. Once bound to something, antibodies can alert immune cells to come and remove the object. Antibodies can also inhibit the object from doing anything nasty, like infecting or damaging a cell. Between species, different types of antibodies have been identified and over the last few decades, scientists have re-engineered this natural system for many different purposes, including medicinal therapy. Recently, researchers have developed a new type of antibody and used it to better understand the activity of a Parkinson’s-associated protein: LRRK2 In today’s post, we will discuss what antibodies are, explore some of the different types that exist, re-examine what LRRK2 is, and review the recent research report. # # # # |
Winter. Source: Sky
Her name is Winter.
And she is a brown coated llama who lives on a research farm near Ghent (Belgium), along with 130 other llamas. You have probably never heard of her, but she has been a critical component in the fight against COVID-19.
Winter (Center, looking left) and friends. Source: Uchicago
Back in 2016, scientists chose a nine-month-old “Winter” as the llama they would inject with spike proteins from SARS-CoV-1 and MERS-CoV viruses, in the hope that she would produce antibodies that could neutralize all coronaviruses.
Note the date – this is why basic research is important to fund.
Jump forward to early 2020, and some of the antibodies that Winter produced back in 2016 were tested on samples of a new coronavirus called SARS-CoV-2 (aka COVID-19). They were found to potently inhibit the virus and Winter’s antibodies appeared in a major research publication:
Title: Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies.
Authors: Wrapp D, De Vlieger D, Corbett KS, Torres GM, Wang N, Van Breedam W, Roose K, van Schie L; VIB-CMB COVID-19 Response Team, Hoffmann M, Pöhlmann S, Graham BS, Callewaert N, Schepens B, Saelens X, McLellan JS.
Journal: Cell. 2020 May 28;181(5):1004-1015.e15.
PMID: 32375025 (This report is OPEN ACCESS if you would like to read it)
This is great, but what do llama antibodies have to do with Parkinson’s?
Llamas are members of the Camelidae family (which include alpacas, dromedaries and camels). And Kevin, their collective noun is unexciting: Herd.
Meet the Camelidaes. Source: Pinterest
One really interesting feature of the Camelidae family is that in addition to having normal antibodies, they also have another class of naturally occurring antibodies that are devoid of light chains.
What?!? Hang on a second. Slow down. What are antibodies??? And what the heck are light chains???
Antibodies are a critical part of our immune system.
When a pathogen (an agent that can causes disease or damage) is detected in your body, there are cells present in our blood system that will inspect it and quickly determine it is ‘self’ (meaning that it is not part of your body) or foreign. Sometime there are objects on the surface of the pathogen that can stimulate an immune system reaction/response.
These are called antigens.
A good example of a pathogen is a virus.
Source: BBC
Once inside the body, the presence of the virus will be detected by cells in the immune system and given that the virus will be presenting antigens on its surface that are clearly not self, an immune response will be initiated. The cells that carry out the immune response are white blood cells known as lymphocytes.
That big cell in the middle is a lymphocyte. Source: ASH
There are basically two types of immune response:
- An antibody response
- A cell-mediated immune response
These processes are carried out by two different types of lymphocyte cells (B cells and T cells). In the antibody response, B cells are activated and they begin to secrete Y-shaped proteins called antibodies.
Antibodies are generally Y-shaped proteins that the immune system naturally and continuously produces to label anything in the body that is ‘not self’ (think of our virus example).
Monoclonal antibodies. Source: Astrazeneca
Antibodies act like alert flags for the immune system. When antibodies bind to something, they alert the immune system to investigate and potentially remove that bound object. In the image below, antibodies (in red) can be seen binding to a virus (in green).
Antibodies binding to a virus. Source: Biology-questions-and-answers
In this fashion, antibodies are a very powerful method of removing or neutralising foreign or dangerous items from the body before they can cause too much trouble.
Antibodies bind to parts of the antigen that are called epitopes.
An epitope is the part of an antigen that is recognised by an antibody. Each antibody targets a specific part of an epitope, while ignoring everything else.
So antibodies bind to a pathogen and cause an immune response?
That is one of their functions.
In addition to alerting the immune system, antibodies by themselves can do a pretty good job of stopping pathogens. For example, they can bind to the pathogen and block it from infecting/damaging a cell by bunging up the mechanism that the pathogen uses to do its nasty work.
Antibody binding to antigens. Source: Venngage
Ok, I understand. But you mentioned ‘light chains’ further up. What is a light chain?
The “immunoglobulin light chain” (by its full name) is a component of an antibody.
As I mentioned above, your typical antibody is Y-shaped in nature. They are composed of two immunoglobulin heavy chains and two immunoglobulin light chains. The two immunoglobulin heavy chains stick together and form the base of the antibody – they are part of the ‘constant region’ of the antibody:
The light chains are in pink. Source: Bitesizebio
Most of the structure of an antibody is constant (always the same), but the top of the Y arms are always variable (or changing from antibody to antibody). These variable parts are the antigen identifying and binding regions, while the bottom stalk of the Y is the part the rest of the immune system interacts with.
Ok, and you said that camels and llamas have normal antibodies as well as antibodies that are devoid of light chains?
Yes.
Members of the Camelidae family (alpacas, dromedaries and camels) have both normal “conventional antibodies” and other antibodies without these light chains (‘heavy chain only antibodies’):
Source: SteyaertLab
And this second kind of antibody is rather unique in the animal kingdom – only sharks and the Camelidae family have them.
I’d rather collect antibodies from llamas. Source: ABCnews
These heavy chain only antibodies have similar potency, specificity and diversity as the conventional antibodies and they have evolved to play an active part in the immune system of these animals.
Interesting. But what is so special about these llama antibodies?
Well, scientists have been developing and engineering antibodies for all kinds of applications – from medicinal to basic research – for decades. In fact, since 1985, almost 100 antibody-based therapies have been approved by medical regulators as drugs and the global therapeutic antibody market was valued at approximately US$115.2 billion in 2018 (Source).
One of the issues with antibodies, however, is their size. Compared to most small molecule drugs, antibodies are huge! In the image below (which is not to scale), you can see that aspirin (on the left) is only 180 daltons in molecular weight, compared to the antibody on the right, which weighs in around 150,000 daltons:
Size matters. Source: Researchgate
As a result, antibodies have a hard time accessing some parts of the body, such as the brain. Antibodies struggle to cross the blood-brain-barrier – a protective membrane that surrounds the brain. It is made up of endothelial cells that are connected by tight junctions. These cells limit the ability of many molecules (particularly large ones) to access the brain, making it a ‘barrier’ for a lot of medications.
The blood-brain-barrier. Source: Bioninja
To get around this problem and to develop more therapeutically useful antibodies, scientists have utilised llama ‘heavy chain only antibodies’ to make nanobodies.
So what are nanobodies?
Nanobodies are a small fragment of the heavy chain only antibodies, consisting of just a single arm of the heavy chain (also called a monomeric variable antibody domain).
The difference between antibodies and nanobodies. Source: SteyaertLab
Nanobodies can be made to be extremely small compared to normal antibodies.
Can they cross the blood brain barrier?
Yes, the first demonstration of this reported in 2012:
Title: Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging.
Authors: Li T, Bourgeois JP, Celli S, Glacial F, Le Sourd AM, Mecheri S, Weksler B, Romero I, Couraud PO, Rougeon F, Lafaye P.
Journal: FASEB J. 2012 Oct;26(10):3969-79.
PMID: 22730440 (This report is OPEN ACCESS if you would like to read it)
Remarkably, in this study, the researchers demonstrated that nanobodies not only easily cross the blood brain barrier, but they also penetrate cells, which increases the potential functional utility.
While a lot of the current medications in the clinic (or going through clinical trial) are based on conventional antibody technology, the future will probably be focused on nanobodies. The intellectual property associated with this area of research is the topic of heated activity for large pharmaceutical companies.
In January 2018, the Belgian biotech company Ablynx was acquired by the Sanofi for $4.8 billion, after rejecting a take over off of $3.1 billion from Novo Nordisk (Source).
At the time, Ablynx had more than 45 nanobody-based therapeutic research programs, with eight of their nanobodies in various stages of clinical trial testing, and their first product (Caplacizumab for the treatment of acquired Thrombotic thrombocytopenic purpura – a rare blood disorder) was approved for clinical use in 2018.
Due to their tiny size and unique structure, nanobodies offer some very important advantages over conventional antibodies – especially when considering their use in therapeutics:
- Nanobodies can be combined with each other (up to 7 in one go), or be combined with other molecules or drugs, allowing for multiple targets to be addressed in one combined drug.
- Nanobodies are small enough to be able to bind with epitopes on targets which are hidden or shielded from the much larger conventional antibodies.
- They are significantly more stable and aggregation resistant compared to conventional antibodies.
- Nanobodies have a larger spectrum of antigenic epitopes, including enzyme active sites.
And it is this last feature that is interesting in terms of our post today.
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# RECAP #1: Antibodies are a key component of our body’s defense system. They act like little red flag binding to and drawing attention to anything that should not be present in our bodies. There are different type of antibodies, but llamas have a unique type that is very small. Researchers have used these to make ‘nanobodies’, which are now expanding the uses of antibodies in research and therapeutics. # |
What do llama nanobodies have to do with Parkinson’s?
Well, recently scientists have been applying these llama-derived nanobodies to Parkinson’s-related research:
Title: Nanobodies as allosteric modulators of Parkinson’s disease-associated LRRK2.
Authors: Singh RK, Soliman A, Guaitoli G, Störmer E, von Zweydorf F, Dal Maso T, Oun A, Van Rillaer L, Schmidt SH, Chatterjee D, David JA, Pardon E, Schwartz TU, Knapp S, Kennedy EJ, Steyaert J, Herberg FW, Kortholt A, Gloeckner CJ, Versées W.
Journal: Proc Natl Acad Sci U S A. 2022 Mar 1;119(9):e2112712119.
PMID: 35217606 (This report is OPEN ACCESS if you would like to read it)
In this study, the researchers were interested in better understanding the function of the protein LRRK2.
What is LRRK2?
Leucine-rich repeat kinase 2 (or LRRK2 – pronounced ‘lark 2’) – also known as ‘Dardarin‘ (from the Basque word “dardara” which means “trembling”) – is an enzyme that has many functions within a cell – from supporting efforts to move things around inside the cell to helping to keep the power on (involved with mitochondrial function).
The many jobs of LRRK2. Source: Researchgate
The LRRK2 gene – the section of DNA that provides the instructions for making LRRK2 protein – is made up of many different regions. Each of those regions is involved with the different functions of the eventual protein. As you can see in the image below, the regions of the LRRK2 gene have a variety of different functions:
The regions and associated functions of the LRRK2 gene. Source: Intechopen
Tiny genetic errors or variations within the LRRK2 gene are recognised as being some of the most common genetic risk factor for Parkinson’s, with regards to increasing ones chances of developing the condition (LRRK2 variants are present in approximately 1-2% of all cases of Parkinson’s).
The structure of Lrrk2 and where various mutations lie. Source: Intech
As the image above suggests, mutations in the PARK8 gene are also associated with Crohn’s disease (Click here and here for more on this) – though that mutation is in a different location to those associated with Parkinson’s. And one particularly common Parkinson’s-associated LRRK2 mutation – called G2019S – is also associated with increased risk of certain types of cancer, especially for hormone-related cancer and breast cancer in women – Click here to read more about this. If you have a G2019S mutation, no reason to panic – but it is good to be aware of this association and have regular check ups.
The G2019S variation (the name designates its location on the gene) is the most common LRRK2 mutations. In certain populations of people it can be found in 40% of people with Parkinson’s (Click here to read more about this).
What is the effect of having the G2019S variation?
If you look at the image above, you will see that this genetic variation sits within the kinase region of the LRRK2 gene.
What does the kinase region do?
A kinase is an enzyme that regulates the biological activity of other proteins. this means that LRRK2 has the ability to regulate the activity of other proteins.
Kinases function by transferring phosphate groups from high-energy, phosphate-donating molecules (like ATP) to specific target proteins – in a process called phosphorylation.
Source: Bmglabtech
Wait. What does any of that mean? What does phos…phory…late mean?
Phosphorylation of a protein is basically the process of turning it on or off – making it useful or inactivating it. From allowing a protein to fold in a particular manner to actually activating/deactivating the function of a protein, phosphorylation is a critical function in cellular biology.
Phosphorylation of a kinase protein. Source: Nature
Phosphorylation occurs via the addition or removal of phosphates. Their addition or removal determines the state of the protein being phosphorylated.
So the kinase region of LRRK2 is important for turning on or turning off other proteins?
In a nut shell, yes.
And am I correct if I assume that the G2019S mutation stops this kinase activity?
No, that would be incorrect.
Rather, quite the opposite.
In the mid 2000s, researchers reported that the G2019S mutation actually increases the kinase activity of LRRK2:
Title: Parkinson’s disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity.
Authors: West AB, Moore DJ, Biskup S, Bugayenko A, Smith WW, Ross CA, Dawson VL, Dawson TM.
Journal: Proc Natl Acad Sci U S A. 2005 Nov 15;102(46):16842-7.
PMID: 16269541 (This report is OPEN ACCESS if you would like to read it)
In this study, the researchers discovered that the G2019S variation did not have any obvious effect on LRRK2 protein levels or localization within cells. But it did cause an increase in the phosphorylation and the autophosphorylation activity of LRRK2.
Autophosphorylation?
LRRK2 can phosphorylate itself. It can regulate its own activity.
Ok. Got it.
This finding led the investigators to conclude that the G2019S variation may result in a ‘gain-of-function’ mechanism that could be influential in the pathology of LRRK2-associated Parkinson’s.
This ‘gain-of-function’ causes trouble by making the LRRK2 protein hyperactive. And in the delicately balanced environment of the interior of a cell, any hyperactive protein is going to cause trouble. Think: Bull in a china shop.
And this has been the dominant thinking in terms of LRRK2-associated Parkinson’s.
What does LRRK2-associated Parkinson’s look like clinically?
People with LRRK2-associated Parkinson’s usually have a good response to levodopa and cannot be distinguished from idiopathic Parkinson’s cases, except that they typically have a slower course of disease progression (there is a lot of variability between cases though).
It there are way to treat LRRK2-associated Parkinson’s?
Not beyond the current symptomatic treatments like L-dopa, but researchers have been working very hard to develop a new class of drugs called LRRK2 inhibitors. These are agents that reduce the hyperactive kinase activity of the LRRK2 protein (Click here to read a previous SoPD post on this topic).
One of the concerns regarding the small molecules being developed as LRRK2 inhibitors is the potential for unintended consequences from long-term use of these agents. We have no idea at present if these drugs will be safe in long-term use.
And this has led researchers to explore more refined and nuanced approaches, in the hope of developing more targeted treatments.
And this brings us back to the llama nanobodies.
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# # RECAP #2: LRRK2 is a protein that functions as a kinase – an enzyme that regulates the biological activity of other proteins. Genetic variations in the LRRK2 gene can result in a hyperactive version of the protein. There is also data indicating that people with spontaneous PD have elevated levels of LRRK2 activity. Biotech companies are developing inhibitors of LRRK2 as potential disease modifying treatments for PD. # # |
So what did the researchers do with LRRK2 and the llama nanobodies?
The investigators took 3 llamas and injected each of them with different fragments of the LRRK2 protein.
The three fragments were from different regions of the LRRK2 protein (specifically the RocCOR, C-terminal subdomain of COR, and the kinase-WD40 regions).
LRRK2. Source: Intechopen
The most prevalent Parkinson’s-associated mutations in LRRK2 are clustered within the RocCOR and kinase domains of the protein, so these were a natural target for the researchers to investigate.
In each case, the injected llama began generating nanobodies to the fragment of LRRK2 protein that the animal had been injected with, and the investigators were able to collect these nanobodies via a small blood sample.
Next, they went through a series of steps to isolate the nanobodies in the blood that were specific for the LRRK2 region in question. This resulted in refined collections of nanobodies targeting the three different fragments of the LRRK2 protein.
Now, it is important to understand that within these refined collections of nanobodies there were hundreds of nanobodies that targeted different regions of the RocCOR, COR, or WD40 fragments. In total, the investigators had 168 different types of nanobodies to further investigate. There was quite a lot of variability between the nanobodies, and the researchers wanted to explore the functional properties of each of them.
The investigators started this characterisation by growing cells in culture that produce high levels of LRRK2. They next exposed the cells in each type of nanobody and analysed the result.
Interestingly, four of the nanobodies robustly reduced the activity of LRRK2. Three other nanobodies reduced the activity of a LRRK2-target protein (RAB10 – a protein that LRRK2 activates), while two additional nanobodies actually increased the activity of that LRRK2-target protein.
To gain a better understanding of the function of these nanobodies (and the LRRK2 protein itself), the researchers mapped out exactly where on the LRRK2 protein each of these nanobodies were binding.
Of particular interest in this analysis is that some of the LRRK2 inhibiting nanobodies bound to regions of LRRK2 that are very different to where the current batch of small molecule LRRK2 inhibitors being developed for Parkinson’s bind to. This is an important detail, because if there are any problems discovered during future long-term clinical trials with the current batch of LRRK2 inhibitors, scientists will have a different approach to immediate employ to reduce LRRK2 activity. Targeting these other regions that the nanobodies are binding to, could provide a different class of LRRK2 inhibition.
These results are rather exciting. The current batch of small molecule LRRK2 inhibitors are blunt tools that do a remarkably good job of inhibiting LRRK2, whereas these nanobodies offer a more refined approach.
Dr. James Beck, chief scientific officer of the Parkinson’s Foundation, offers an interesting insight to this new research:
“Before, scientists could only turn the LRRK2 protein on or off. However, these results allow for the precise modulation of LRRK2 activity. This is like moving from a stereo that only had an on-off switch to one that now has a full suite of controls to fine-tune the sound” (Source)
So what does it all mean?
One of the exciting areas of disease modification research for Parkinson’s has been the development of LRRK2 inhibitors. These are molecules that have been carefully engineered to reduce a hyperactive version of the LRRK2 protein, and to test a fundamental idea in our theory of how LRRK2-associated Parkinson’s progresses. Over the next few years, this new class of drugs will start to be clinically tested for their long-term safety and efficacy.
As with all new classes of drugs, nothing is assured and anything could potentially happen. To date, the first of these agents to be clinically tested has demonstrated satisfactory levels of safety and tolerability. But that therapy has only been tested in Phase I studies for up to 1 month of treatment. LRRK2 inhibitors will need to be tested for 12 months+ to determine if they are changing the course of the condition. If long term use of any of these drugs is in any way problematic, we need to have alternatives ready to go.
One potential alternative could come from research into Llama-derived nanobodies.
It is important to appreciate that this new research is very early days in its development, and that these nanobodies should currently only be viewed as research tools that are helping us to better understand the biology of LRRK2. But they could be employed to develop the next generation of LRRK2 inhibitors for Parkinson’s, which will offer unique and potentially more personalised approaches to differentially modulate the activity of this protein.
Certainly something to keep an eye on.
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Winter is not the only llama that deserves mention in the fight against COVID.
This is Wally:
Wally the llama. Source: timesofisrael
And antibodies from Wally have also been found to be potent inhibitors of COVID-19 and its variants:
Title: Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2.
Authors: Xiang Y, Nambulli S, Xiao Z, Liu H, Sang Z, Duprex WP, Schneidman-Duhovny D, Zhang C, Shi Y.
Journal: Science. 2020 Dec 18;370(6523):1479-1484.
PMID: 33154108 (This report is OPEN ACCESS if you would like to read it)
This video introduces you to Wally and his impact on the world:
I didn’t want Wally not to get mentioned and to be left out.
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