Optimisation of the manufacture of a homogeneous synthetic haemoglobin as a novel Oxygen Therapeutic / Blood Substitute

Blood transfusion is a lifesaving technology; however, allogeneic red blood cells have inherent disadvantages such as limited shelf life, need for blood group typing, and they cannot be used in immune compromised individuals and those that reject transfusions due to religious reasons. The historical use of HIV- and hepatitis-contaminated blood still shows ramifications today through public inquiries and the ongoing health issues of surviving patients. Blood products are much safer today, with globally around 108 million units of red blood cells transfused annually in an industry worth >$10Bn. However, new blood borne diseases are discovered on a regular basis with the potential of permeating the transfusion population before detection. Additionally, the need for blood group typing and the short shelf life limits the ability to stockpile the supplies necessary for major disasters. It also precludes routine transfusion support in ambulances and in population centres distant from major hospitals (including use by the military).

Haemoglobin (Hb) is the protein in the red blood cell that carries oxygen from the lungs to the tissues. Therefore, a synthetic Haemoglobin-based oxygen carrier (HBOC) at first glance appears to be an ideal starting point for a safer alternative to erythrocyte transfusions. However, clinical trials of HBOCs have typically seen poor outcomes. More recently HBOCs have been promoted as an oxygen 'bridge' for patients promising the possibility of a therapy that takes oxygen to the tissues even though the circulation is failing, thereby protecting or maintaining vital organs. This may be targeted to patients with medical conditions undergoing surgery, for example brain injuries, heart surgery, kidney transplantation and shock following blood loss resulting from trauma injury. Additionally, HBOCs may be used for oxygenation of solid tumours to enhance therapies, treatments for sepsis, sickle cell disease, haemolytic anaemia and many more. Nonetheless concerns over the safety and efficacy of HBOCs remain.

There is a growing realisation that the inherent toxicity of HBOCs, relating to nitric oxide (NO) scavenging and oxidative damage, may largely be responsible for the poor clinical outcomes. While some focus has been given to tackling NO scavenging, very little research has focused on the issue of oxidative damage from 'rogue' enzymatic activities of HBOCs. At Essex we have an expert understanding of the mechanisms and consequences of the damaging oxidative reactions of Hb. Through previous MRC DPFS funding we have successfully developed a genetically engineered HBOC based on the more stable foetal Hb that addresses the inherent toxicities of cell-free Hb. This is the first HBOC to tackle both issues of NO scavenging and oxidative damage utilising recombinant technologies to re-engineer the fundamental properties of the Hb molecule. Our novel mutant Hb addresses -and resolves- the inherent toxicities of cell-free Hb that caused adverse side effects in previous generations of HBOC. Coating the surface of the HBOC with polyethylene glycol (PEG) extends the lifespan of the HBOC in blood, increases its viscosity and decreases its immunogenicity. We have developed a uniquely uniform system of PEGylation that, unlike previous techniques, does not detrimentally effect oxygen binding and cooperativity.

The purpose of this award is to follow up on the successful development of our uniquely stable and unreactive HBOC product to provide a route to clinical manufacture of the product. This development will be to the clinically required standards and will seek to overcome the previous issues associated with protein production. In addition, we will conduct tests designed to show safety and efficacy of the product and to provide evidence that our product has overcome the major issues of previous generations of HBOC and hence provide confidence of positive outcomes in a full program of preclinical and clinical trials.

There is significant clinical need for a synthetic oxygen therapeutic / blood substitute that is both long-lasting and sterile. As haemoglobin (Hb) is the body's natural oxygen carrier the use of cell-free Hb is an ideal starting material for such an agent. However, such Haemoglobin-Based Oxygen Carriers (HBOCs) display an inherent capacity to induce cytotoxicity through oxidative reactions, causing cell and tissue damage. They also scavenge nitric oxide (NO) leading to hypertensive effects.

Through previous MRC funding, we successfully developed an engineered HBOC that overcome the limitations and toxicities of previous generations. The addition of our patented, genetically engineered 'through protein electron pathways' enhances the capacity of plasma reductants to prevent cytotoxic oxidative reactions. In addition, mutations that decrease NO scavenging have been added, avoiding the heme loss and complement activation that has compromised a previous recombinantly produced HBOC with this property.

The focused aim of this current project is to use the expertise developed in our previous MRC DPFS-funded success by developing a manufacturing pathway to clinical trials for our product. Meeting the exacting GMP standards required by regulators is essential. Working with a technology and innovation centre (CPI) we will develop a scalable GMP-compliant manufacturing process. We will utilise our novel engineered system of PEGylation (patent filed) that overcomes the issue of heterogenous PEGylation seen with previous methodologies. This has the added unique benefit of not interfering with O2 binding or cooperativity. Finally, we will show that the previous issues with HBOCS have been addressed through demonstration of product quality, safety and efficacy in animal models, that will help attract future funding for a full program of preclinical and clinical trials.

Commercial and Clinical Benefit.
The success of the project would impact on clinical applications that aim to protect or maintain the functional integrity of vital organs at risk due to medical conditions and/or surgical procedures. Trauma represents a significant potential market opportunity for using an HBOC as an oxygen therapeutic agent to treat acute ischemic complications and improve patient outcome measures. Market research studies have estimated that there are >60 million trauma cases per year in the US and the EU. Of these trauma admissions ~2-2.5 million require transfusion of RBCs. This represent the ideal target patient population who exhibit sufficient tissue ischemia with oxygen debt to benefit from treatment with an HBOC. HBOCs are expected to make a significant impact on the acute market due to their ability to provide immediate treatment without the need to carry different blood types or undertake blood typing before administration. Furthermore, if an HBOC is able to also demonstrate even a small reduction in mortality in a trauma resuscitation setting, then the clinical benefit would be extensive.
Clinical impact could also include other ischaemic conditions including: heart (myocardial infarction, cardiac arrest, angioplasty, bypass surgery), brain (stroke, traumatic brain injury), kidney (transplantation), spinal cord (vascular surgery), and gut (surgery, shock). Additional applications may be applied to oncology to enhance oxygenation of solid tumours (to enhance radiotherapy, chemotherapy and immunotherapy); organ transplantation (ex vivo perfusion to prolong storage hold-time for heart, lung, kidney, or liver); sickle cell disease (acute vaso-occlusive crisis); sepsis; acute haemolytic anaemia.
Average NHS costs for a treatment of a patient in ICU is >£1900 per day in England and Wales whereas cost per day on an average general hospital ward ~£400 [2016 data, www.wales.nhs.uk/documents/delivery-plan-for-the-critically-ill.pdf]. Therefore, using products like our HBOC that, if they could successfully treat severe tissue ischemia and shorten ICU stay by a day, or decrease the duration of hospital stay by 1-2 days can have significant economic impact on NHS costs.

Research and Professional Skills
The project would benefit the professional career development of the post-doctoral research assistant and researcher Co-I. Due to the nature of this project the research assistant and RCo-I will also be encouraged to develop their entrepreneurial skills, interact with the subcontractors and advisors and learning process development. The research assistant will be trained in a wide range of research skills, including gaining scientific practical skills in research and biotechnology, communication skills through the dissemination of the research results at meetings and in writing articles describing the project outcomes for scientific journals. As well as courses organised by the University's Research and Enterprise Office, one-to-one advice will be available from the advisers to the project.
Many of the skills are transferable to other employment sectors. Transferable skills include data handling and analysis, independent planning and communication of results. The post-doctoral research assistant (Essex) and Researcher Co-I (UCL) will be made aware of the RCUK Concordat to support the career development of researchers and will also be encouraged to take advantage of the processional development opportunities offered by the Department (e.g. Essex's and UCL's Professional Development and Training Units/ Career Development and Training).