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In July 2020, ProMIS Neurosciences, Inc. (TSX:PMN.TO) announced that it had entered into a revenue-sharing joint venture with BC Neuroimmunology Lab (BCNI) to develop a blood-based diagnostic for Alzheimer’s Disease (AD). This agreement builds on a previous arrangement with BCNI to develop an antibody test for COVID-19. Both of the efforts will use surface plasmon resonance (SPR) systems which provide real-time, label-free and blood-based analysis. They provide a cost-effective approach that allows for monitoring of analyte kinetics not possible with other systems and are particularly amenable to neurodegenerative disease screening. With improved understanding of the implication of several biomarkers, including pTau and neurofilament light chain (NfL), new tests are being used to screen, monitor and diagnose neurodegenerative disease providing tools critical to advancing therapies.
Since the discovery of predictive biomolecular analytes, biosensors have been used to detect and quantify biological targets of interest and are widely used in healthcare. Biosensors are also employed in environmental monitoring for soil, food and water quality management. In the life sciences space, biomedical measurement analytes include enzymes, cells, metabolites, antibodies and other biological material. Biosensor devices can operate using electromagnetic, electrochemical, optical, piezoelectric and thermometric methods to monitor real-time, highly specific, biomolecular recognition events. The sensors typically include a transducer, a data processing system and a biological element that interacts with the intended analyte being tested, yielding a broad array of critical information.
The modern use of sensors can be traced back to as early as 1906 when the German scientist Max Cremer demonstrated that acid concentration in a liquid correlated to electric potential between it and fluid located on the other side of a glass membrane. Soon after, the concept of pH was introduced. Within the next decade, Edward Griffin and J.M. Nelson demonstrated enzyme immobilization with invertase on aluminum hydroxide and charcoal substrate. In the 1960s, the father of biosensors, Leland C. Clark, Jr, invented the oxygen (Clark) electrode (1). Fast forward to today, biosensors are integral to a multitude of technologies, such as surface chemistry, microfabrication, microfluidics, nanomaterials, electrochemical sensors, field-effect transistors, optical sensors and acoustic sensors. Configurations include lateral flow systems and lab-on-chip devices. These varied technologies and configurations are aimed at sensing an equally diverse set of analytes. Advances in fabrication and nanotechnology also enable increasingly compact designs.
Sensors used in SPR consist of a glass surface coated with an inert metal, usually gold due to its non-reactive and non-oxidative features (2). On top of the metal coating, a self-assembled monolayer is deposited, providing the linker layer to the metal surface. The interactants used to bind to the analyte can be either electrostatically or covalently attached to the exterior of the slide, with the latter favored due to its stronger bond.
Lateral Flow Assay Biosensor Featuring Antibodies (3)
SPR as a mechanism for conducting molecular binding studies was first described in an academic paper by Rothenhäuslar and Knoll (4) in 1988 and began to be used widely by pharmaceutical companies and research universities in the 1990s. The instruments most widely used during this first decade of availability used Pharmacia’s BIAcore platform that became increasingly sensitive and specialized as successive generations were developed. The SPR technology relies on a beam of incident light passing through a prism then reflecting off of a thin metal coating on the sensor. The reflected light will shift its angle due to the excitation of surface plasmons and produce a dark band known as the resonance, or SPR angle, on the detector. The angle of the refracted light is sensitive to molecular binding on the metal coating as surface plasmon activity causes a dip in the intensity of reflected light on the detecting surface (5). This is due to the refractive index of the prism changing based on the accumulated mass near the metal surface on the sensor. This phenomenon enables the investigator to measure the binding of analytes to ligands that are attached to a sensor surface. Binding affinities can also be measured over time as the analyte flows over the ligand generating an SPR response (association constant) which decays (disassociation constant) when the analyte injection ends.
The Resonance of Surface Plasmons
The majority of an atom’s volume is comprised of the electron cloud, with the outermost electrons loosely bound and subject to ‘deformation’, analogous to a fluid medium that can oscillate. A surface consisting of electron gas density can oscillate or ‘ripple’ like a pond in what are called surface plasmons. Surface plasmons that are excited at the interface between a metal layer and a sample reflect changes on the sensing surface. This is represented in a Kretschmann configuration by the illumination of a gold surface where its reflection is captured by a detector. The reflection contains information about plasmon activity and changes in plasmon activity that correlate to binding activity on the sensor. In particular, where light incidence generates plasmons, light will not be reflected at the resonance angle and there will be a dark band in the reflection across this region. Changes in this absorption pattern signify changes in plasmon characteristics, which can be a sign of activity on the surface, such as when a ligand is bound. Evidence of ligand binding implies the presence of an analyte in a solution, such as a biomarker in blood.
Kretschmann Configuration of SPR Detection (6)
SPR offers numerous advantages compared to other diagnostic testing approaches. It can detect a wide variety of targets, from small molecules to biologics and ions to viruses. They are also specific, sensitive and do not require labeling and provide a quantitative indication of the avidity or affinity of the binding. Higher throughput and specificity and the ability to reuse the sensor make SPR a highly efficient approach in diagnostic testing. A summary of the key benefits includes:
‣ Label free detection allows analyte identification without labelling reagents & protocols
‣ Small sample sizes are sufficient for binding kinetic experiments
‣ Reusable sensor chips reduce the need for consumables
‣ Consistent results are generated
‣ Real time monitoring of biomolecule interaction and kinetics
‣ Technique able to analyze complex, unpurified samples
Improved understanding of key biomarkers has provided new, noninvasive ways to measure the earliest, asymptomatic stages of neurodegenerative disease. Some of the most commonly used biomarkers correlated to changes in cognitive health and AD include neurofilament light chain (NfL) and multiple isoforms of phosphorylated tau (pTau): 181, 217 and 231. These biomarkers have gained prominence as the definition of AD has shifted more towards a biological one. In early 2018, the FDA expanded what it recognized as acceptable endpoints in AD trials and would allow the use of response and monitoring biomarkers for the disease. The combination of new biomarkers and acceptance of their use in pivotal trials opens the doors for a more efficient approach to developing a therapy for AD.
Neurofilament light chain (NfL)
In previous articles we have discussed ProMIS’ use of NfL, which can provide a quick determination of whether or not neuronal damage is occurring. Neurofilament light chain is a neuronal cytoplasmic protein. Neurofilaments are about 10 nm in diameter and are abundant in axons and are also present in perikarya and dendrites. The axon’s cytoskeleton is composed of neurofilaments as well as other structures. It is these neurofilaments, specifically the 68 kDa light chain, that are found in blood plasma as a result of neuroaxonal damage, are known to correlate with progression of AD.
Axonal Cytoskeleton (7)
In a paper published July 2019, Mattsson et al. investigated the correlation between longitudinal plasma neurofilament light (NfL) and Alzheimer’s disease (AD). Using gold standard methods of measurement, Aβ and tau cerebrospinal fluid (CSF) biomarkers and multiple imaging measures, with clinical diagnosis and cognitive testing, the authors found that plasma NfL was correlated with AD. This finding suggested that the cytoskeletal components of neurons could be used to noninvasively track progression of the disease (8). 1583 participants were included, and of those, 25% had no cognitive impairment, 54% had mild impairment, and 21% had AD dementia. NfL levels in the subgroups were 32.1 ng/L, 37.9 ng/L and 45.9 ng/L, respectively, all highly statistically significant (9). The trend in NfL levels also correlated with CSF biomarkers Aβ42, total tau and phosphorylated tau levels at significant levels (10).
NfL is represented by the cytoskeletal proteins that remain after the destruction of neurons. The marker appears in cerebrospinal fluid (CSF) or the blood and its concentration is stable over time and indicates neurodegenerative diseases ranging from AD to MSA. SPR is able to detect even small quantities of the neuronal cytoplasmic protein. The new biomarker offers the potential for a cost-effective, convenient and objective method of blood-based detection and monitoring of AD progression, starting as early as 20 years before cognitive decline. Early detection can provide new opportunities for intervention, slowing and perhaps even stopping the disease. The joint venture between ProMIS and BCNI will focus first on a blood-based assay for NfL and p-tau181.
Neurofilament Subunits (11)
Phosphorylated Tau (pTau)
In a collection of recent work, phosphorylated tau (pTau) has been shown to have an association with AD. Tau plays a critical role in microtubule stabilization, which is key to the structure of neurons and malfunctions as a result of phosphorylation. The phosphorylation affects the protein’s ability to stabilize microtubules, and promotes tau aggregation into neuronal filaments observed in many neurodegenerative diseases. Tau is phosphorylated when a phosphoryl group (PO3) binds to the side chains or residues of the tau protein, specifically at serine, threonine and tyrosine phosphorylation sites. Without Tau, the microtubules become unstable, resulting in neuronal death.
Mechanism of Tau Malfunction and Subsequent Neuronal Death (12)
pTau 181 is tau that has been phosphorylated at threonine 181. In pivotal work done by Karikari et al., pTau 181 was found to be a highly specific biomarker for AD. In succession, a discovery cohort of 37, first validation cohort of 226 (TRIAD), second validation cohort of 763 (BioFINDER-2) and a primary care cohort of 105 generated a total of 1131 enrolled patients in the broader study. Across all of the cohorts, pTau 181 gradually increased with AD severity. Younger and healthier older adults had low concentrations, older, amyloid-beta positive asymptomatic adults had higher and the amyloid-beta positive MCI and AD groups had the highest concentrations of pTau 181 in their blood plasma. pTau 181 plasma concentration had a predictive ability of AD that was significant at the 0.1% level versus all other groups. Diagnostics are also able to distinguish among neurodegenerative disorders using pTau 181 for frontotemporal dementia, vascular dementia, progressive supranuclear palsy or corticobasal syndrome, Parkinson’s disease and multiple systems atrophy.
ProMIS has highlighted pTau 181 as one of the key pTau isoforms that it will measure in its collaboration with BCNI along with NfL. The appearance of pTau 181 early in the disease provides a valuable window of opportunity for detection and prevention.
ProMIS plans to build a portfolio of assays that enable early detection, which may introduce other isoforms of pTau to the stable of diagnostics offered. Studies have been conducted demonstrating that several forms of pTau are effective biomarkers for AD and predictive of cognitive decline (13). pTau 181 has shown the ability to predict both tau and amyloid-β pathologies and isolate AD from other neurodegenerative diseases. The Karikari study showed that concentration of pTau 181 was lowest in young, healthy adults and highest in amyloid β-positive mild cognitive impairment (MCI) and Alzheimer disease groups. The 217 isoform of pTau was also shown to provide advance notice of AD approximately 20 years before symptoms (14). Other work found that pTau 217 and pTau 181 are highly correlated to each other. pTau 231 has also provided evidence of its utility providing overall greater specificity than pTau 181 for detecting AD (15).
Collaboration for the Future
While ProMIS and BCNI have only recently joined forces, both ProMIS’s expertise in identifying unique epitopes for misfolded proteins implicated in neurodegenerative disease and BCNI’s expertise in SPR-based biosensing create the foundation for exciting and effective technologies in the battle against AD. BCNI’s status as a private, full-service neuroimmunology lab at the University of British Columbia Vancouver hospital with experience in Surface Plasmon Resonance (SPR) and other assays provides a solid foundation for test development. Future advancement of this collaboration is expected and may incorporate ProMIS’ proprietary peptide antigens and expand into testing for other neurodegenerative diseases such as amyotrophic lateral sclerosis, multiple system atrophy and frontotemporal dementia among others.
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2. Noble metals besides gold that may be considered are silver, copper, platinum and palladium among others.
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9. Significant at the 0.1% level.
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