Site Directed Inactivation of Biological Agents

Although microbes and humans have co-evolved, some bugs continue to pose a threat to humans. The introduction of sanitation and supply of clean drinking water has had and continues to have a huge impact on the transmission of many diseases caused by a range of microorganisms. Over the past two centuries, vaccination has also provided a very efficient cost effective protection against many pathogens. In the past century the discovery and development of antibiotic and anti-viral drugs has provided yet more protection. However over the past three decades we have witnessed the emergence of diseases that have crossed over from an animal reservoir and adapted to infect humans often with dire outcomes, notably, HIV, SAR's and a range of influenza viruses with avian or swine origins. These are natural emerging infectious diseases, each requiring considerable resources to manage, understand and to develop countermeasures. More recently another threat has emerged, the "deliberate releases" - the intentional spread of a biological or chemical agent, such as might occur in a biowarfare or bioterrorist incident. Microbes that can be weaponised to be dispersed and infect major metropolitan areas via a range of delivery mechanism, some are non-contagious, others are extremely contagious. Super-imposed on this deliberate use and release of microbes is the potential ability to genetically engineer and incorporate additional factors to make them more potent, such as resistance to known drugs, greater infectivity, the induction of debilitating host factors (cytokine storm). In some cases vaccinations is not an option, since though effective, the time required to achieve protection is too long (i.e., anthrax vaccination). An immediate fast acting molecule/intervention is required.
We are faced with two challenges, to create new drug countermeasures and decontamination procedures. In this proposal, we have devised a strategy for rapidly creating molecules that are highly specific for a target pathogen, and critically can deliver a knock-out punch to either kill the microbe or inactivate its toxins. The key essence is to create molecules that do not naturally exist in nature, but the components bits actually do and to figure out a way of putting them together so that each part still functions.
Antibodies are unique highly specific molecules that can be generated by a number of approaches. Moreover accessing the genes that encode the antibodies is relatively simple. Antibodies have a modular structures, the binding site of an antibody is made up of two variable domains held together in the correct orientation by additional constant domains. It is this pairing that confers the ability to recognise targets. With current technology it is possible to make antibodies to almost any biological and many chemical targets. However, binding to a target confers specificity, but that may not be enough to kill or inactivate a target.
Hydrogen peroxide, is a reactive oxygen species (ROS) and is a potent bactericidal agent that is used in most homes as a general disinfectant at low levels, but it does not discriminate between good and bad microbes. What if we could harness the specificity of the antibody binding site and the ability of molecules that generate ROS to target high levels of ROS directly on the pathogen surface?
A protein derived from jellyfish called KillerRed generates ROS in response to illumination with light of a certain wavelength. Secondly glucose oxidase (used in diabetic glucose sensors) generates ROS in the process of enzymatically degrading glucose. Both of these proteins are potent generators of ROS. We propose to create a hybrid molecule that is part antibody binding site and part ROS generator and to evaluate these as anti-microbial agents. This will be achieved by inserting the ROS generating proteins between the antibody binding domains to retain and stabilise the functions of the two proteins.

The objective of this proposal is to investigate the specific delivery of molecules that can generate reactive oxygen species (ROS) at a microbial target surface. We will use a modular design, which permits interchange of the specificity domains (i.e., antibody VH and VL domains) and the effector domains (fluorescent proteins mRFP/KillerRed or glucose oxidase). Over the past 2 years we have developed and validated a novel way of assembling recombinant single chain antibodies in which we have substituted the widely used flexible (Gly4Ser)3 linker with a more precise rigid molecule that spans the same distance. Such linkers stabilise the VH/VL pairing and do not facilitate dissociation and aggregation. Secondly these rigid molecules consist of proteins with defined functions, in the first example, monomeric fluorescent red protein. Such novel fusion molecules retain the binding properties of the antibody and the fluorescence properties of the RFP. Such molecular "docking" can also be carried out with other protein modules that have the correct spatial alignment of the N- and C- termini. RFP, KillerRed both produce reactive oxygen species (ROS) when illuminated by light within the visible spectrum, thus the activity can readily be switched "on" and "off" by simple illumination. A more potent producer of ROS is glucose oxidase and this may also be docked in as a linker. This approach may be further extended to incorporate other additional protein effectors. In this proof of concept study we will investigate the targeted inactivation of a protein (associated with a known biothreat agent), a bacterial cell (that is a surrogate, but has potential for developing a commercially viable therapeutic application) and a eukaryotic cell (as a surrogate for an infected cell, but also has potential for developing a diagnostic/therapeutic application) via oxidation.

Who might benefit from this research?
DSTL will benefit from having access to a generic platform for rapidly developing diagnostic and therapeutic molecules to defined biothreat agents.
Also biopharmaceutical companies, research institutes and academic researchers (globally) may adopt the approach for rapid target validation. It may also provide insight into possible novel therapeutic targets, and potentially the corresponding biologic drug, in particular for surface accessible sites. The results would contribute to firmly establishing the UK as a leader in the field. The general public would benefit in the long term by the improved diagnostic and therapeutic option in a range of indications leading to enhanced health outcomes.

How might they benefit?
This is a new approach to tackling difficult problems identified in the infectious disease area. It will provide a platform technology that can be integrated with existing antibody discovery platforms, hybridoma, phage or ribosome display, since the VH and VL sequences can be interchanged to confer the desired specificity. If an antibody molecule inhibits a particular pathogenic process, it will be possible to follow it and to modulate it. As we better understand the molecular markers that differentiate good cells from bad cell it will provide opportunities for linking diagnosis directly to the treatment.

Enterprise & innovation:
The patent on the AFP technology has already been filed in the US (20110268661 FLUORESCENT FUSION POLYPEPTIDES AND METHODS OF USE Nov 3 2011) and three leading global antibody reagent supply companies are in discussions for licensing this technology, thus the building block technology is already providing a wealth creation opportunity. It is a very powerful screening tool for the biopharmaceutical industry. The technology may also provide opportunities for developing novel diagnostic platforms that could attract further R&D investment leading to commercialisation and exploitation. With better diagnostics comes earlier detection which leads to earlier interventions and more beneficial outcomes, which significantly enhance the quality of life. If we get the targets right, we will be able to deliver on both detection and treatment.