Team:UCL/Description

Inspiration Solution Encapsulins Modularity Cargo Manufacturing

Project Inspiration and Description

Inspiration

Currently there is a lot of interest in drug delivery systems (DDS) in order to reduce the side effects of conventional cancer treatments such as chemotherapy, and increase treatment efficiency. However, most of the developed DDS up to date tend to have one or more of these five major disadvantages: toxicity, immunogenicity, low drug carrying capacity, ability to release cargo on demand or high production cost (1). Through our research into DDS, we discovered Bacterial Microcompartments (BMCs) which naturally hold enzymatic proteins and localise metabolic reactions. Recent research in the field has genetically modified BMCs to widen their applicability in biotechnology. Inspired by their robust ability for compartmentalisation and versatility in their applications, we sought to investigate how we can utilise protein-based compartments to address current issues with DDS and create a modular drug delivery platform in an elegant and effective manner.

Our Solution

Encapsulin coated with DARPins and loaded with cargo illustration
Figure: The modular encapsulin-based framework design. Two different types of encapsulins of different sizes were investigated ( T. maritima and M. xanthus). The surface of the encapsulin was coated with different types of proteins (targeting DARPins and the small fluroescent protein iLOV). The encapsulin was loaded with sfGFP and toxic photosensitizers, and the components and complex were produced both in vivo and in vitro.

We developed a modular drug delivery platform based on self-assembling bacterial nanocompartments, called encapsulins. To functionalise these delivery vesicles, we fused them with Designed Ankyrin Repeat Proteins (DARPins) – monoclonal antibody mimics that can be expressed in bacterial hosts (2). When attached to an encapsulin, DARPins will facilitate their internalisation by selective receptor mediated endocytosis. Finally, the encapsulins were loaded with cytotoxic cargo that will interrupt cellular processes once internalised. As a proof of concept, we tested the system on SK-BR-3 breast cancer cells, with DARPin929 binding to the overexpressed HER2 receptors.

Key Characteristics of Encapsulins for Drug Delivery

The encapsulins used for our delivery system are from the bacteria Thermotoga maritima and Myxococcus xanthus. Protein sequences can be fused both onto the outer and the inner surfaces of the nanocompartments (3) and proteinaceous cargo can be encapsulated via the hydrophobic interactions of short targeting sequences (4, 5). Unlike other mainstream vectors, such as virus like particles, encapsulins are pH-resistant, thermostable and are non-immunogenic (6). Importantly, one of the determinants in our decision of encapsulins over other carriers, it their ability to self-assemble in vivo and in vitro without further proteolytic or chemical processing.

Modularity and Specificity

We selected DARPins as our binding agents hoping that the existing DARPin libraries (with over 350 targets) and ease of creating a new DARPin via genetic fusion (2, 7), will widen the applicability of this platform to target different receptors on different cell lines. Their consistent structure ensures that specificity properties will remain unaffected when one DARPin is exchanged for another. In addition, DARPins have the highest affinity of all synthetic binding protein types, being able to detect targets as low as 5–100 pM in concentration, potentially allowing higher potency and allowing subsequently smaller effective concentration to achieve a required clinical outcome. Finally, their thermodynamic stability pairs well with encapsulins, enabling the use of heat purification (2).

Cytotoxic Cargo

Based on input from industry professional Michael O’Neill, physician Dr Yin Wu and academic Dr Andrew Care we have decided to use encapsulins with cargo that would provide a second degree of selectivity to unspecific drug non-target effects. We are loading our encapsulins with photosensitisers – fluorescent proteins that produce chemical changes in another molecule once induced by light of certain wavelength. In our case, photosensitiser miniSOG produces reactive oxygen species that mediate cellular toxicity (8). By directing light only on the tumour area, we are able to provide localised treatment thus minimising potential side effects to healthy cells, ensuring improved safety over currently available treatments.

Manufacturing

Successful biotherapeutics must meet three major requirements: high end-product quality, economic viability, and accessibility to the public. Therefore, large-scale manufacturing platforms which allow robust and cost-effective production must be developed.

The biological complexity of monoclonal antibodies used in current targeted therapies requires the use of organisms which can achieve the required post-translational modifications for correct folding and function of these proteins. Therefore, adherent mammalian cells are often employed. Although efficient mammalian-based production systems have been developed, yields are considerably smaller than prokaryotic-based system due to the slower growth rates and inherently complex biological requirements (e.g. multiple growth factors) required for their growth. Furthermore, their adherent nature means that these cells need a surface for attachment and Multi-tray planar T-flasks. These static culture conditions result in heterogeneities, low surface-area-to-volume ratio and a lack of online control systems, thus rendering optimisation for large-scale production more challenging.

In comparison, bacterial expression systems are well established, require simpler and cheaper growth media and can achieve higher cell densities in a shorter period of time. These cells are usually grown in suspension cultures, which allow better homogeneity, control, and better use of the volume provided for growth. The existing product heritage additionally means that existing industrial manufacturing footprint can be used. All these attributes make bacterial fermentation usually more attractive to investors for use in biotherapeutics production.

In these terms, our approach towards targeted delivery is more advantageous. Encapsulin nanocompartments and DARPins have been proved to be easily and successfully produced in bacteria, specifically Escherichia coli. Combining this with well characterised large-scale industrial bioprocesses means that we could produce a successful new class of biotherapeutics with improved consistency and scalability while minimising their manufacturing cost, which is required in the modern competitive healthcare market.

References

  1. Giessen TW. Encapsulins: microbial nanocompartments with applications in biomedicine, nanobiotechnology and materials science. Synth Biol Synth Biomol. 1 de octubre de 2016;34:1-10.
  2. Plückthun A. Designed Ankyrin Repeat Proteins (DARPins): Binding Proteins for Research, Diagnostics, and Therapy. Annu Rev Pharmacol Toxicol. 6th January 2015;55(1):489-511.
  3. Lagoutte P, Mignon C, Stadthagen G, Potisopon S, Donnat S, Mast J et al. Simultaneous surface display and cargo loading of encapsulin nanocompartments and their use for rational vaccine design. Vaccine. 2018;36(25):3622-3628.
  4. Sutter M, Boehringer D, Gutmann S, Günther S, Prangishvili D, Loessner M et al. Structural basis of enzyme encapsulation into a bacterial nanocompartment. Nature Structural & Molecular Biology. 2008;15(9):939-947.
  5. Nichols RJ, Cassidy-Amstutz C, Chaijarasphong T, Savage DF. Encapsulins: molecular biology of the shell. Crit Rev Biochem Mol Biol. 3 de septiembre de 2017;52(5):583-94.
  6. Heinhorst S, Cannon GC. A new, leaner and meaner bacterial organelle. Nat Struct Amp Mol Biol. 1 de septiembre de 2008;15:897.
  7. Using Mimics to Get Around Antibodies’ Limitations [Internet]. The Scientist Magazine®. at: https://www.the-scientist.com/lab-tools/using-mimics-to-get-around-antibodies-limitations-64264
  8. Dolmans D, Fukumura D, Jain R. Photodynamic therapy for cancer. Nature Reviews Cancer. 2003;3(5):380-387.