by Vivian Yang
art by Qingyang Meng
Among the array of vials and test tubes adorning Professor Raymond Hamer’s lab bench at the Free University in Brussels, a remarkable discovery waiting to be uncovered was circulating within, of all things, a sample of camel’s blood. It was not until Hamers gave a couple of his biology students this blood for an assignment that the hidden wonders within the sample were finally unearthed. The assignment? Analyzing a key component found within the camel’s immune system, an antibody [1,2].
Camels, humans, and almost all mammals have antibodies. These are proteins that play a vital role in the adaptive immune system– the specialized part of the immune system that works to attack and destroy foreign invaders– by recognizing or removing pathogens, like viruses or bacteria [3]. Hamers’ students were tasked with separating the antibodies they found into two main parts: a pair of heavy protein chains and a pair of light protein chains. Both heavy chains, which are essentially longer and larger in weight than light chains, link to form a Y shape. On the top part of the "Y" shape (the "V"), each heavy chain is flanked on the outside by a light chain [4]. Think of a fork with prongs that you would use to stab food.
Each section of an antibody serves a different task, but the variable domains at the very tips of the V-shaped “arms” of the Y are involved with binding to antigens, or the substance on foreign pathogens like a virus or bacteria that the immune system recognizes. This is because variable domains are where binding sites of the antibody are found; the structure of these binding sites varies from antibody to antibody [3]. Acting as a “sensor”, variable domains can latch onto a part of the antigen, called an epitope, via their complementary shape that triggers an immune response [5]. This snug fit is similar to that of a key and a lock. This understanding of antibodies has been accepted as true for decades, so Hamers and his students were quite taken aback when they came across some very unprecedented results.
Much to their surprise, Hamers’ students could only detect heavy chains, and no light chains in the antibodies circulating within the camel’s blood. Had they made a mistake? Perplexed, Professor Hamers repeated the experiment with fresh blood and found that the students were right, camels produce antibodies with just heavy chains [6].
A little over three decades later, these strange antibodies have been a continual focal point for scientific inquiry [7]. As more of their unorthodox characteristics began to surface, researchers began to investigate whether the hidden wonders within camelids may present promising breakthroughs in both research and clinical applications, especially when tackling some of the most formidable diseases in the brain, such as neurodegenerative diseases and brain cancers.
A Unique Structure: Small and Condensed
These camelid antibodies, referred to as heavy-chain-only antibodies (HCAbs) are found within the entire Camelidae family, which includes not only camels but also alpacas and llamas. HCAbs in the camelid immune system have various unique structural elements that distinguish them from the conventional antibodies found in humans. For one, the absence of light protein chains results in a molecular weight that is approximately 40% less than that of the most common type of antibody in humans [1]. Like conventional antibodies, HCAbs contain variable domains that act as the business end of the antibody. Conventional antibodies consist of four variable domains in total, formed by two pairs: one pair from the heavy chain and one from the light chain. HCAbs only have one pair from the heavy chains [5].
When observing these variable domains in isolation from the entire HCAb, their molecular weights are less than conventional antibodies by almost tenfold, earning the “nanobody” title [8]. To avoid confusion, these minuscule antigen-binding fragments at the ends of camelid heavy-chain-only antibodies will be referred to as nanobodies (or Nbs) throughout this article. So how do scientists extract these nanobodies from camelids? The advancement in scientific technology has facilitated the identification and production of antigen-specific nanobodies derived from HCAbs in camelids [3]. A camelid is first injected with a target antigen, which causes an immune response: the camelid develops an antibody to protect against the target antigen. Scientists can then take this genetic information to make the nanobody portion of the HCAb from the camelid's immune cells and put it into E. coli bacteria. After being produced by the bacteria, the nanobody can be extracted and purified [9].
Significance: A More Advantageous Alternative
What was it about these nanobodies that caused such a stir? To understand the novelty of these antibody fragments, we must first take a step back and examine the use of monoclonal antibodies (mAbs), which share many functions and purposes with nanobodies and therefore provide an apt basis for comparison. mAbs are manufactured proteins made with copies of antibodies from the body to mimic how antibodies naturally work in the immune system. They are highly valued for their high specificity to a single epitope, aiding in their ability to reach specific targets to treat diseases including cancer and inflammation [10]. As German scientist Paul Ehrlich proposed, antibodies act as magic bullets, flying straight to their targets and (if functioning correctly) avoiding anything else in their path [11]. What
makes these “bullets” magical is their incredible specificity for a single target [12]. However, there have been many critics calling into question the accuracy of Ehrlich’s metaphor.
Although they are often tools of choice, mAbs still hold many limitations that nanobodies have the potential to overcome. While mAbs are often costly and time-consuming to produce, nanobodies consist of much simpler structures that are easier to modify and manufacture [8]. Additionally, nanobodies can conform to and recognize a broader scope of epitopes with their small size and convex shape. The antigen binding sites of nanobodies can therefore lodge into concave sections on target molecules that are usually inaccessible to conventionally large mAbs. Unlike conventional antibodies that have heavy and light chains, each with a triplet of CDRs (complementarity-determining regions), which are responsible for determining antigen specificity, the HCAb only has one triplet of CDR on the sole heavy chain. Yet with this limited number of CDRs, it has one loop of CDR that is slightly longer than the corresponding loop on the conventional antibody. This longer loop can access more surfaces for antibody-antigen contact; think of this loop as an extended magnet that pulls in molecules efficiently with its outstretched reach [7,13].
Perhaps the most obvious, but significant advantage of nanobodies over mAbs, is their small and compact size. mAbs are very large and complex proteins; this consequently limits mAbs’ ability to reach antigens when it is unable to penetrate or disperse enough through certain tissues in the body [14,15]. While mAbs weigh about 150 kDa (kilodaltons, a unit of molecular mass) with a variable domain weighing about 50 kDa, nanobodies– the variable domains of heavy chain only antibodies in camelids– weigh merely ~12-15 kDA [14]. The inherently smaller size of nanobodies leads to improved tissue penetration, allowing for more specific and efficient drug delivery. More specifically, nanobodies have shown a higher potential of crossing a persistent obstacle faced by mAbs: the blood-brain barrier [7].
The Blood-Brain Barrier (BBB): Brilliant but Burdensome
Barrier structures are integral in protecting organs in the human body by preventing certain molecules from entering including the blood-testis barrier, blood-eye barrier, etc. Of immense clinical focus is the integrity of the blood-brain barrier (BBB), which safeguards the central nervous system (CNS) [5]. Acting as a highly selective filter that regulates the substances entering the brain, the BBB is an extensive network of different cell types and blood capillaries that is vital in the protection of the CNS from infections and toxic substances [16]. Antibodies carry out their functions by binding to target molecules, so they need to be delivered into the brain to have functionality [5]. The BBB, however, prevents the entry of many larger-sized molecules such as antibodies [15].
Therefore nanobodies, with a size that is 10x smaller than normal antibodies, have a much higher chance of penetrating the BBB [5]. This could lead to profound solutions in tackling devastating diseases that afflict the brain, such as neurodegeneration and brain tumors.
Neurodegenerative Disease: Alzheimer’s and Parkinson’s
Neurodegenerative diseases are characterized by damage and destruction to parts of the nervous system over time, especially within the brain. The most common of these diseases, Alzheimer’s disease (AD) accounts for dementia in an estimated 6.9 million Americans aged 65 and older, and this number could grow to 13.8 million by 2060 [17]. The aggregation, or accumulation, of the protein tau and amyloid-beta, are two closely intertwined theories that pinpoint the causes behind AD progression in the brain. In individuals with AD, these proteins no longer fold normally, becoming misshapen, and transition into amyloid fibrils, or long thread-like structures of protein that spread from cell to cell (Habicht). The abnormal clumping and tangling of proteins such as tau and amyloid-beta are some of the most popular hypotheses explaining neuronal death in AD [14]. Detecting or neutralizing these proteins before accumulating and becoming misshapen through antibody targeting would be major steps towards the treatment of neurodegenerative diseases, a foremost goal of researchers worldwide.
The initial enthusiasm over employing mAbs to achieve this objective eventually subsided when many of these mAbs did not exhibit much success. While a few mAbs have demonstrated promising results, a handful failed clinical trials as there were no differences observed in reduced cognitive decline or clearance of aggregated protein when compared to placebo groups. One mAb that did work, called “aducanumab”, upon repeated injections and high dosages triggered unwanted immune responses and consequently adverse side effects. Essentially, the body produced antibodies against the therapeutic mAb. Nanobodies, however, have demonstrated a much lower risk of producing unwanted immune reactions and therefore may outperform mAbs [18].
Researchers at the University of California, Los Angeles, were just one of many intrigued by the peculiar proteins in camelids as they tackled the ability of misfolded and aggregated tau protein to trigger normal tau proteins to conform toxically as well, initiating a chain reaction of misfolded protein. They developed engineered nanobodies to target tau aggregation while also overcoming the limitations associated with conventional antibodies. They did this by producing a panel of nanobody inhibitors that bind to specific, structural parts of tau proteins responsible for driving protein aggregation and spread. This successful reduction in tau may be a step closer towards a treatment/inhibitor for Alzheimer's disease [19].
Another study took a more preventative approach to amyloid fibril formation by harnessing nanobodies to inhibit the formation of fibrils in the protofibrils stage. During this stage, protein fibrils increase in length and thickness before forming mature protein fibrils. Due to the difference in conformation states between native proteins and amyloid fibrils, the research team saw an opportunity to discriminate between states, employing a nanobody that specifically targets protofibrils. This nanobody, named B10, stabilizes protofibrils before they can form into mature fibrils, which makes them more likely to be broken down by the cell [20].
The second most common neurodegenerative condition is Parkinson’s disease (PD), with a global prevalence that increased from 2.5 million to 6.1 million between 1990 and 2016 [21]. PD is classified as a progressive disorder that is caused by the degeneration of nerve cells in a part of the brain called the substantia nigra, which controls movement. As these nerve cells die off or become impaired, they lose their ability to produce one of the most important chemicals in the brain, dopamine. Dopamine operates in a fine balance with other neurotransmitters to help coordinate millions of nerve and muscle cells involved in movement. Without dopamine, this balance is disrupted, triggering the hallmark symptoms of Parkinson’s: slowed movements, tremors, balance problems, and more [22].
PD is characterized by the aggregation of α-synuclein (α-syn), designating it as part of a group of neurodegenerative diseases called synucleinopathies. Like tau and amyloid-beta, α-synuclein can accumulate to form fibrils which in turn form toxic, dense spherical clumps of protein known as Lewy bodies. These Lewy bodies cause neurons to stop interacting with neighboring neurons and eventually die [22].
mAbs for PD are currently underway and, like those for AD, have experienced successes and failures [18] [Tsitokana]. Researchers at John Hopkins University, however, saw the value of kickstarting the emerging usage of nanobodies in PD and wielding their unique advantages to achieve the same goals as mAbs. They conducted a groundbreaking study in which they were able to produce a nanobody– deemed PFFNB2– to specifically target clumps of misshapen α-syn protein, referred to as α-syn preformed fibrils (PFF), over the regular α-syn protein. In their research, they were able to demonstrate the nanobody's ability to penetrate brain cells and untangle the α-syn protein structures causing harm. Furthermore, it impeded the α-syn assembly and reproduction in mice. This nanobody could potentially halt the progression of PD and hold therapeutic promise in treating other related synucleinopathies [23].
Another study with the same mission of targeting the α-syn protein harnessed a nanobody with a different tactic. Researchers at New York University focused on the degradation of α-syn rather than the inhibition of its aggregation. They developed a nanobody-based protein degrader that was designed to enhance cells’ natural protein breakdown in α-syn. By promoting the breakdown of α-syn, researchers observed improved clearance of α-syn in both primary cell cultures and mouse models, making their nanobody-based protein degrader a viable candidate for synucleinopathy, or diseases associated with α-syn aggregation [24].
Brain Tumors: Tackling Glioblastoma
While neurodegenerative diseases wreak havoc on the brain by deteriorating cells, cancer, characterized by uncontrolled growth and spread of cells, can also tear down brain tissue and lead to premature death. Glioma refers to a broad category of brain tumors that arise from mutated glial cells, which support neurons in the brain. Glioblastoma (GB) is the most aggressive type of glioma. Despite intense treatment protocols, most patients develop resistance to therapy, resulting in an overall median survival rate of less than two years [25]. This therapeutic failure can be attributed to the presence of glioblastoma stem cells that are resistant to chemotherapy and radiotherapy [26].
One study investigated the usage of nanobodies to inhibit key antigens involved in glioma progression. Although glioma starts in glial cells, it can rapidly spread to other areas of the brain and spinal cord [27]. They first identified eight novel antigens associated with GB and examined the effects of four nanobodies on glioma cells, discovering two nanobodies– Nb225 (anti-TUFM) and Nb79 (anti-vimentin)– that displayed promising tumor suppressive effects, such as inhibition of cell growth and migration, with no harm to the non-cancerous cells that protect and repair neuronal damage [26]. A subsequent study later focused on the protein TRIM28 which displayed high expression in GB and is associated with the progression of GB. The research team constructed a nanobody, Nb337, that selectively targets TRIM28 and was found to significantly inhibit cell growth and GB stem cell spread in zebrafish brains. These results also suggest that stem cells could be blocked from invading or spreading the cancer to other parts of the body [25].
Limitations: A Ways To Go
Nanobodies have received significant attention and praise for the seemingly endless opportunities they present in combating some of the most challenging diseases inflicted on the human brain. Despite the promise shown in a multitude of studies, it is important to recognize the limitations in the usage of nanobodies that could dampen their efficacy.
In order for researchers and clinicians to utilize nanobodies to visualize and measure biological processes at the cellular level, an application known as molecular imaging, the organisms must be modified. For example, in some cases, a nanobody must be tagged with a chemical compound that allows for radiometal labeling, which involves attaching radiometals to biomolecules to track movement through cells or reactions. Consequently, such modifications can alter certain properties of nanobodies such as size or structure, which could affect the performance of the nanobody [28]. One study explored the passage of nanobodies through the BBB and although they were able to demonstrate efficient entry into the brain through the BBB, their data also suggested rapid clearance of these nanobodies after entry; this is due to their low molecular weight. Consequently, the nanobody’s ability to accumulate an effective level within the CNS can be heavily impeded, which will impact their therapeutic properties [29].
Conclusion
Nanobodies are still largely in preclinical stages, requiring more research to fully optimize the usage of nanobodies in treating diseases in the brain. Nonetheless, nanobodies present exciting possibilities in the future as more effective alternatives to conventional antibodies in tackling the hidden, unreachable regions in the brain affected by disease. There has been persistent evidence suggesting that the “magic bullet” metaphor used to describe monoclonal antibodies may be misleading as there are still many obstacles such as the BBB and shortcomings such as complicated, large structures hindering mAbs’ effectiveness. Nanobodies, however, could bring us a couple of steps closer to bringing this mythical magical bullet to life, equipping us to more effectively fight complex diseases in the brain.
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