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Discovery of a DARPin-based antitoxin against α-cobratoxin

Stewart, Trenton (2020) BION03 20192
Degree Projects in Biology
Abstract
Snakebite envenomations has for a long time been an overlooked problem, despite the hundreds of thousands of deaths and disabilities caused by this disease each year. However, since snakebite envenoming predominantly affects the rural areas of third-world countries, there was an absence of research funding and drive to address this profound concern. Consequently, costly and often highly immunogenic animal-derived antivenoms still constitute the sole treatment option for snakebite envenomations.

Fortunately, public interest and funding has increased in the last decade, thereby, presenting a new horizon for snakebite therapeutics. This has led to a shift in how researchers approach the treatment of envenomations. It has been proposed... (More)
Snakebite envenomations has for a long time been an overlooked problem, despite the hundreds of thousands of deaths and disabilities caused by this disease each year. However, since snakebite envenoming predominantly affects the rural areas of third-world countries, there was an absence of research funding and drive to address this profound concern. Consequently, costly and often highly immunogenic animal-derived antivenoms still constitute the sole treatment option for snakebite envenomations.

Fortunately, public interest and funding has increased in the last decade, thereby, presenting a new horizon for snakebite therapeutics. This has led to a shift in how researchers approach the treatment of envenomations. It has been proposed that, instead of targeting the whole venom, one could focus on only neutralising the clinically relevant toxins within the venom. Moreover, the advancement in antibody research has led to novel approaches towards treating snakebite envenomings, such as the utilization of human monoclonal immunoglobulin G antibodies (IgGs). Whilst IgGs present a thoroughly investigated and highly reliable therapeutic scaffold, its rather large size (150 kDa) could present hurdles in neutralizing certain toxins quickly (especially in deep tissue) and the associated manufacturing costs could also complicate its global deployment to the poorer regions of the globe.

Small non-antibody-scaffolds present a promising alternative, since they are likely to overcome some of the key limitations of IgGs, while retaining most of their benefits. Particularly, designed ankyrin repeat proteins (DARPins) carry significant promise due to their impressive safety profile in humans, high thermostability, low manufacturing cost, and high engineerability.

Therefore, this thesis aimed to establish the capability of DARPins to bind with high-affinity to α-cobratoxin from monocled Cobra (Naja Kaouthia). To achieve this, the power of the phage display technique was harnessed to screen a DARPin-library to discover α-cobratoxin-specific binders. Following four panning rounds, the outputs were evaluated via plate tests and polyclonal ELISAs. Unfortunately, numerous issues arose throughout this thesis, complicating and slowing down the selection process. In the end, no α-cobratoxin-specific DARPin binders were discovered and there was no time to repeat the selection process. Nevertheless, key issues have been identified and solutions have been proposed to ensure that future selection campaigns will hopefully be efficient and successful.

Indeed, whilst antibody discovery can be complicated, time-consuming, and often relies on a certain amount of luck, I have no doubt that my first steps towards a first-ever snake toxin binding DARPin will aid in the endeavour of harnessing the potential of this scaffold in the context of next-generation antivenoms. Hopefully, this will eventually lead to a substantial improvement in the treatment of snakebite evenomations and help alleviate the global health burden that is snakebite. (Less)
Popular Abstract
Come to the DARP-side: attempted discovery of a DARPin-based antitoxin against α-cobratoxin

Snakebite envenomation is a global problem that results in over 400,000 permanent disabilities and between 81,000 and 138,000 deaths each year. However, these numbers likely represent a significant underestimation of the true health impact of snakebites. The problem is further substantiated by inefficient serum-based antivenoms constituting the only specific treatment for the past 120 years.

Fortunately, technological advances, alongside an increase in public funding, are now presenting a new horizon for antivenoms. Hence, researchers are exploring novel approaches towards the treatment of evenomations, such as the use of small protein... (More)
Come to the DARP-side: attempted discovery of a DARPin-based antitoxin against α-cobratoxin

Snakebite envenomation is a global problem that results in over 400,000 permanent disabilities and between 81,000 and 138,000 deaths each year. However, these numbers likely represent a significant underestimation of the true health impact of snakebites. The problem is further substantiated by inefficient serum-based antivenoms constituting the only specific treatment for the past 120 years.

Fortunately, technological advances, alongside an increase in public funding, are now presenting a new horizon for antivenoms. Hence, researchers are exploring novel approaches towards the treatment of evenomations, such as the use of small protein scaffolds as a prospective antivenom. One such scaffold, is designed from natural ankyrin repeat proteins and is involved in numerous protein-protein interactions in almost all known species. These designed ankyrin repeat proteins (DARPins) carry significant promise as an antivenom, which is attributed to their small size (aiding in rapid distribution and tissue penetration), high thermostability (don’t require cold storage), ease of production, and no reported adverse reactions in humans.

Thus, we aimed to establish the capability of DARPins to bind with α-cobratoxin from the monocled cobra (Naja kaouthia). α -cobratoxin is the most abundant toxin in the venom of this species and is known to induce muscle paralysis, leading to respiratory failure.

Phage display technology was utilized to provide the capability of discovering α-cobratoxin-specific DARPin binders. Unfortunately, numerous issues arose throughout this thesis, complicating and slowing down the phage display process. In the end, no α-cobratoxin-specific DARPin binders were discovered and there was no time to repeat the project. Nevertheless, key issues and potential solutions were identified to try and ensure that future campaigns will be efficient and successful.

The discovery of toxin-binders can be complicated, time-consuming, and often relies on a certain amount of luck. However, the initial steps were taken in this thesis that will hopefully lead to the first-ever snake toxin binding DARPin. With a bit of luck, this will eventually lead to a substantial improvement in the treatment of snakebite evenomations and help alleviate the global health burden that is snakebites.

Master’s Degree Project in Biology, 60 credits, 2020


Technical University of Denmark
Department of Biotechnology and Biomedicine
Advisor: Andreas H. Laustsen

Lund University
Department of Biology
Advisor: Lars Råberg (Less)
Please use this url to cite or link to this publication:
author
Stewart, Trenton
supervisor
organization
course
BION03 20192
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9028282
date added to LUP
2020-09-08 14:20:34
date last changed
2020-09-08 14:20:34
@misc{9028282,
  abstract     = {Snakebite envenomations has for a long time been an overlooked problem, despite the hundreds of thousands of deaths and disabilities caused by this disease each year. However, since snakebite envenoming predominantly affects the rural areas of third-world countries, there was an absence of research funding and drive to address this profound concern. Consequently, costly and often highly immunogenic animal-derived antivenoms still constitute the sole treatment option for snakebite envenomations.

Fortunately, public interest and funding has increased in the last decade, thereby, presenting a new horizon for snakebite therapeutics. This has led to a shift in how researchers approach the treatment of envenomations. It has been proposed that, instead of targeting the whole venom, one could focus on only neutralising the clinically relevant toxins within the venom. Moreover, the advancement in antibody research has led to novel approaches towards treating snakebite envenomings, such as the utilization of human monoclonal immunoglobulin G antibodies (IgGs). Whilst IgGs present a thoroughly investigated and highly reliable therapeutic scaffold, its rather large size (150 kDa) could present hurdles in neutralizing certain toxins quickly (especially in deep tissue) and the associated manufacturing costs could also complicate its global deployment to the poorer regions of the globe. 

Small non-antibody-scaffolds present a promising alternative, since they are likely to overcome some of the key limitations of IgGs, while retaining most of their benefits. Particularly, designed ankyrin repeat proteins (DARPins) carry significant promise due to their impressive safety profile in humans, high thermostability, low manufacturing cost, and high engineerability. 

Therefore, this thesis aimed to establish the capability of DARPins to bind with high-affinity to α-cobratoxin from monocled Cobra (Naja Kaouthia). To achieve this, the power of the phage display technique was harnessed to screen a DARPin-library to discover α-cobratoxin-specific binders. Following four panning rounds, the outputs were evaluated via plate tests and polyclonal ELISAs. Unfortunately, numerous issues arose throughout this thesis, complicating and slowing down the selection process. In the end, no α-cobratoxin-specific DARPin binders were discovered and there was no time to repeat the selection process. Nevertheless, key issues have been identified and solutions have been proposed to ensure that future selection campaigns will hopefully be efficient and successful. 

Indeed, whilst antibody discovery can be complicated, time-consuming, and often relies on a certain amount of luck, I have no doubt that my first steps towards a first-ever snake toxin binding DARPin will aid in the endeavour of harnessing the potential of this scaffold in the context of next-generation antivenoms. Hopefully, this will eventually lead to a substantial improvement in the treatment of snakebite evenomations and help alleviate the global health burden that is snakebite.},
  author       = {Stewart, Trenton},
  language     = {eng},
  note         = {Student Paper},
  title        = {Discovery of a DARPin-based antitoxin against α-cobratoxin},
  year         = {2020},
}