Cell Guidance Systems' mission is to improve the delivery of cancer immunotherapeutic drugs, focussing on late-stage prostate cancer, a disease which kills 12,000 men each year in the UK. The body's immune system can recognize and destroy cancerous cells. However, cancers gradually develop mechanisms to evade the immune system, ultimately undergoing uncontrolled growth. In the last three decades, major progress in cancer treatment has been made using immunotherapies that enable the immune system to control cancer. Cytotoxic T lymphocytes (CTL, a kind of white blood cell) are a major focus of immunotherapies. They are the front-line immune cells actively engaging with cancer cells. CTLs can recognize and bind to cancer cells, killing them with lytic enzymes. However, CTLs often become depleted and exhausted in tumours. Many cancer immunotherapies work by restoring functional CTLs. This immunotherapeutic strategy has allowed some late-stage cancer patients to achieve durable remission. However, only subsets of defined cancer types are responsive and, because the drugs also reach normal tissues, side effects are often severe. Improved targeting of drugs to improve the anti-cancer functionality of CTLs in tumours has the potential to increase drug efficacy whilst reducing side effects and is urgently needed. To improve drug delivery, we have been exploring the utility of mononuclear phagocytic immune cells (MPs) such as monocytes, macrophages and dendritic cells. MPs are found in the blood and ingest large particles. They also home towards, and then actively infiltrate cancer, accounting for as many as 50% of the cells present. This suggested to us the possibility of using MPs to actively, and more efficiently, deliver microscopic drug particles into the tumour to activate CTLs. We have developed microscopic, slow-release protein drug particles, called PODS, which are ingested by MPs. We have shown that cytokine protein drugs contained in the PODS are sustainably released from the MPs in a potent, bioactive form. We have also shown that when PODS are injected intravenously into a mouse with cancer, the cancer responds to the treatment. the number of CTLs increases and, consistent with low off-target toxicity, the drug is undetectable in the serum. This drug delivery strategy has enormous potential for improving cancer immunotherapeutic drug delivery and enabling new treatment options for all solid cancers. In the present project, we plan to use PODS-delivered chemokines to recruit CTLs and dendritic cells into prostate cancer and activate these using PODS-delivered cytokines and immune checkpoint inhibitors.

Purified recombinant proteins are widely used in research, medicine, and industry. Cytokines, hormones and antibodies have been important in reshaping the field of medicine over the last half-century. How these proteins are manufactured, stored and delivered to their site of action (e.g. diseased tissue) is fundamental to their utility. We have developed PODS, a nature-mimetic technology, that adds value to all aspects of a recombinant protein's life cycle. PODS are sub-micron scale cubic protein co-crystals (containing a cargo protein) produced in insect cells. PODS utilizes an innovative manufacturing technology, based on a process that naturally occurs in silkworms, to produce bioactive proteins incorporated into sub-micron scale protective crystals (made of a bio-inert polyhedrin protein). The crystals greatly simplify the purification of the recombinant protein, as they are physically distinct from other components of the production cell. Storage instability is addressed as cargo proteins are stabilized within the crystal lattice and have high levels of stability over many months, even at elevated temperatures in aqueous suspension. Finally, following administration, at the site of action, the crystals are degraded by proteases providing a matrix-degradation dissolution-based cargo release system. This provides sustained release of the soluble cargo protein over a one-month period. We have developed PODS for research and therapeutic applications, primarily for localized sustained release of cytokines. Most recently, we have demonstrated that PODS are able to harness phagocytic immune cells, such as monocytes, neutrophils and macrophages, to target the delivery of immunostimulatory cytokine proteins to cancer. We plan to extend this delivery approach to other protein classes, particularly peptides and small antibodies, known as nanobodies. However, the packaging efficiency of cargo proteins into PODS, constituting around 1% of the total protein, is a limitation: Whilst this is sufficient for delivering highly potent proteins such as cytokines, other protein classes, such as antibodies and anti-microbial peptides, require higher loading efficiency. In addition to therapeutic applications, there are industrial applications of PODS, such as the biomanufacturing of cultured meat, in which cost reductions enabled by increased cargo density would be highly advantageous. The project will focus on the bioengineering of PODS to generate a novel PODS architecture with increased cargo density. Two independent strategies will be assessed to create PODS that have a higher cargo protein loading capacity. These PODS will be tested in a range of assays to assess cargo packing density, release profile and bioactivity.

World meat consumption has tripled since 1970 and will increase a further 76% by 2050\. In the future, there will not be enough meat available for the world's population. This shortage will hit low-and-middle-income countries, where meat is an important but limited source of nutrient-dense protein, vitamins and minerals, especially hard. Over 80 billion animals are slaughtered annually for meat, the majority being factory-farmed. Increasing livestock production isn't the answer as this promotes climate change, environmental destruction and infectious disease spread. Livestock farming generates 15% of human-made greenhouse gas (GHG) and will contribute 0.5°C to global temperatures if continued. Cattle-ranching and animal-feed crops also account for most agricultural water use and 85% of rainforest clearance. Overcrowding and poor welfare standards help spread diseases, including swine and avian flu, and are major contributors to human food poisoning. Excessive livestock antibiotic use is fueling increases in antibiotic-resistant bacteria which render antibiotic medication useless: Alarmingly, antibiotic-resistant pathogens are forecast to cause greater mortality than cancer by 2050\. Cultivated meat (CM) grows animal cells in bioreactors to produce a product similar to conventional meat but without the need for any animal suffering. CM will also use fewer resources (energy, land and water) and produces less GHG, counteracting environmental issues. Since CM only requires a few cells from animals, it eliminates farming welfare issues and antibiotic use. CM, which appeals to consumers considerate of these issues, is undertaken in carefully controlled, sterile conditions vastly improving food safety. The global US$246.9Mn CM market is set to increase to $6.8Bn by 2030\. However, to achieve this forecast, this new approach needs to produce meat at a scale before it can then address future meat shortages. The first CM burger cost $330,000, demonstrating edible CM products are possible albeit at very high costs. The challenge is to make CM in large amounts, using a cost-effective and market-competitive process. Millions of tons of meat are consumed annually, so this will ultimately necessitate the development of massive (\>10,000L) bioreactors capable of generating very high-density cell cultures. This requires cells capable of growing under demanding conditions and carefully balancing nutrients and cell-toxic by-products. These nutrients (such as growth factors) need to be cheap, well-characterised and perform consistently. This project combines the skills and capabilities of three UK universities and four UK companies developing livestock cell lines, recombinant protein technologies, hydrogels and bioreactor components to collaboratively develop technological solutions for CM production.

The mechanism by which a drug travels following administration (e.g. by intravenous injection) to reach its molecular target is critical to efficacy. Innovations in drug delivery have been fundamental to many medical advances. Most recently, RNA drugs, including widely used vaccines against SARS-CoV-2, have entered the market. RNA drugs required novel mechanisms of drug delivery which overcame specific challenges: Whilst conventional drugs act outside cells or are small enough to permeate through to intracellular targets, RNA drugs are only effective inside cells, and their large size necessitates a specific delivery mechanism to carry them across the cell wall and into the cell cytosol where they act. Viruses and lipid nanoparticles (LNPs, resembling small hollow spheres of fat) are used to deliver currently approved RNA drugs. Viruses and LNPs envelop and protect the RNA from damage whilst outside the cell and are able to dock or fuse with the cell wall to make the final delivery of RNA. However, these technologies have significant shortcomings including production costs, batch consistency, storage stability, immunogenicity, lack-of-targeting, the efficiency of cell entry, and endosomal escape. Whilst improvements in these technologies are being made, a fresh approach may better address these challenges. Particles access cells through a variety of passive and active mechanisms. Phagocytosis is a process in which cells actively ingest particles (most efficiently in the 0.3 - 5 microns size range). This process is particularly efficient in professional mononuclear phagocytic (MP) cells. These immune cells present vaccine antigens, ingest debris and eliminate invading pathogens. They also migrate to diseased and injured tissue and are involved in a variety of processes, including the generation of vaccine immunity, autoimmune diseases, wound healing and cancer. Some MPs, such as the liver's Kupfer cells, are not of haemopoietic origin. MPs also harbour many important pathogens. RNA drugs that effectively target MPs have the potential to impact many devastating diseases. We have engineered microscopic protein crystal particles, called PODS crystals, that contain specific bioactive protein cargos. MPs ingest PODS crystals and secrete their cargo proteins intact. We have also shown that cargo proteins enter the cytosol - the area of the cell where RNA is active - suggesting the possibility to deliver RNA. Here, we plan to generate protein crystal particles to deliver RNA to MP cells. Once this has been demonstrated, we will test RNAs' ability to modify MP cells' behaviour across a range of biomedical applications.

Innovative growth factor functionalized products for the lifescience research market

To embed underpinning knowledge of biomaterials and capability in hydrogel engineering, to enable development of bioinks, 3D matrices and functionalised microcarriers for incorporation into and enhancement of our Polyhedrin Delivery System sustained release protein technology.

"Parkinson's disease (PD) is a neurodegenerative disorder that affects around 1% of individuals over the age of 55\. The disease is associated with loss of a relatively small number of cells, called dopaminergic neurons (DNs), which are located deep in the centre of the brain. PD is a progressively debilitating disease with patients currently treated using drugs and therapies to reduce the severity of the symptoms. However, none of the available therapies impact the overall progression of the disease. Therefore, there is an urgent, unmet clinical need to develop a therapy which is able to slow-down or, ideally, reverse the progression of PD. Strong evidence from animal models shows that regenerating DNs can arrest PD progression. The most effective way to promote DN survival is by treatment with neurotrophic growth factors (nGFs) (naturally occurring signalling proteins which are vital for the development and the maintenance of the healthy nervous tissue). nGFs are highly potent molecules which, if deployed systemically (i.e. intravenous), have marked potential for toxicity, causing damage to healthy non-target cells. Consequently, nGFs need to be precisely administered by surgery. nGFs are fragile proteins with very short half-lives, typically of _minutes to several hours_. However, any nGF drug needs to be active for _weeks to months_ in order to have a measurable effect on DNs. Since repeated surgery is impractical, a great focus of research has been the development of technologies and devices to stabilize, and/or provide sustained release of nGFs from a depot which can be surgically delivered to the required location, deep in the brain. We are developing PODS (POlyhedrin Delivery System), a recently developed sustained-release protein technology based on a natural system that evolved in an insect virus lifecycle. By engineering this system, PODS is able to neatly package and protect perfectly formed nGFs inside protein crystals. These protein crystals are highly stable but start to loosen and release their valuable cargo in contact with proteases from living cells. The rate of cargo protein release can be controlled over time, and release over several months has been achieved. We have demonstrated the utility of PODS using rat models of disease. PODS has the unparalleled potential to deliver on the promise of nGFs to provide vital disease-modifying therapy to treat PD. In this project, we plan to evaluate this potential in various PODS crystal formulations containing nGFs in cell-based and small animal models of PD."

Awaiting Public Project Summary

Osteoarthritis (OA) is a huge problem to the affected individual and the healthcare system. In the UK, 29% of adults over 45 have OA, of which 9% is severe (Arthritis Research UK) – equating to 6.5m people. It can cause severe pain in joints, for example the hip and the knee where and can cause great difficulty walking. No current treatment exists for OA with most patients needing a joint replacement. Early in the process, defects can occur in the surface of the joint, the cartilage which contribute to the developmemnt of OA. This study aims to develop a new way of repairing and regenerating cartilage defects. We will use proteins, called growth factors, that are known to help cartilage healing, and will surround them in a new covering inspired by nature, called a polyhedron protein packages (PODS). PODS allows the molecule to be released slowly which we hope will make them better able to heal the cartilage. The anticipated outcome of this project will be to provide vital data on the how effective the new treatment is. This will allow investment to be sought to make these treatments in large amounts and move to the first trials in people with OA.

The majority of vaccines have a narrow temperature window within which they must be stored to maintain functionality. The development of vaccines that are stable at ambient temperatures is a major challenge in their delivery to the people that need them the most, in low-income settings where the refrigeration infrastructure required is poor. We have developed an innovative vaccine manufacturing system, based on a patented protein production mechanism. This system exploits the unique properties of the polyhedrin protein naturally expressed by the silkworm (Bombyx mori) cypovirus. The polyhedrin protein forms large, temperature-stable and pH-stable crystals within infected insect cells. These crystals attach to, and subsequently encase, mature cypovirus virions. This results in enhanced stability and viability of the virion, leading to a markedly longer window of infectivity. Genetic engineering techniques have been used to adapt this viral survival mechanism to encapsulate any given tagged recombinant proteins by co-expression with polyhedrin protein. This in-vitro insect cell expression system is known as PODS (POlyhedra Delivery System). PODS provides key advantages for vaccine production, including (1) rapid development, to address emerging viruses, (2) scalability of manufacture, and (3) inherent stability of cargo protein structure, even over extended periods in warm and wet conditions, eliminating the need for cold-chain supply and the need for frequently repeated, more costly manufacturing schedules. The aim of this study is to develop a vaccine delivery platform based on PODS. The project will have 3 streams: 1) Develop PODS encoding and encapsulating glycoproteins from a range of priority pathogens with pandemic potential and confirm temperature stability of PODS vaccines over an extended time. 2) Test PODS for their ability to induce neutralising antibodies. 3) As a proof of concept, test PODS vaccines for their ability to protect against infection. We will examine three viral infections based on prioritization by UKVRDN and based on our expertise: (1) Zika virus, an emerging pathogen linked to malformation of the brain in children of mothers infected during pregnancy. (2) Ebola virus, a viral hemorrhagic fever that has caused the death of >11,000 individuals in West Africa since December 2013. (3) Lassa fever, an acute viral haemorrhagic illness that is endemic in the rodent population in parts of West Africa which causes illness in 300,000 individuals each year with around 5,000 deaths as a result. By the end of these initial studies, we aim to obtain proof of concept that the PODS platform can be used to generate temperature stable vaccines capable of inducing a potent neutralising antibody response. Having shown the efficacy of the vaccine we plan to move to GMP production of PODS in a phase II application.

Cell Guidance Systems is planning to develop a protein purification technology which has potential to significantly impact the way proteins are both manufactured and used for cell research and disease therapy. The technology is significantly differentiated from alternative protein manufacturing technologies and addresses very significant, multi £bn research and therapeutic markets. We are seeking support for an IP review.

The manufacture of cells in a controlled and reproducible manner is a critical challenge in regenerative medicine. We will develop an integrated platform technology for the rapid identification of novel, efficient stem cell expansion and differentiation protocols that are optimised for translation to clinical grade manufacturing. Several innovative technologies will be combined to perform high throughput GMP compatible screens to discover novel cell production protocols. These protocols will be transferred to a scaled-up bioprocess where a novel cell imaging technology will be utilised to monitor cell performance. Successful protocols will be translated to a GMP manufacturing process for final validation. In this way, safe, robust, cost-effective GMP validated protocols will be rapidly discovered, greatly reducing the cost and time for development of regenerative cell therapeutics.

Nature of Issue: Significant challenges are associated with the current provision of blood transfusion services including (1) Recruiting an adequate pool of donors to provide reliable, matching supply of donor to recipient types (2) Typing of collected material (3) Testing for pathogens (4) Cell counting (5) Storage, inventory control and logistics to recipient (6) Disposal of unused blood. For transplants, in comparison with bone marrow, there are many advantages to using cord blood, including reduced requirement for matching between donor and recipient, and superior engraftment. These advantages have lead to a rapid increase in the use of cord blood in childhood leukaemia. However, cord blood volumes are small, with limited numbers of cells. Consequently, 90% of patients (with higher body mass), are not able to benefit from this therapy. CD34+ expansion can increase the number of cells in a cord blood sample generating a therapeutic dose for patients regardless of body mass. Similarly, there are significant potential benefits for using erythrocyte expansion. Several groups in the UK have received funding to develop methods to manufacture red blood cells. \this includes a Scottish consortium which recently received a £2.5m grant from Scottish Development which in addition to a £3m grant from the Wellcome Trust (ref 1). Although technically feasible, growth factor costs for CD34+ expansion technology are prohibitively expensive. For example, Delaney et al (Nat Med, 16:232(2010) describe an improved process for making CD34+ cells which requires growth factors costing > £140,000. These growth factor costs, rather than technical barriers, are widely acknowledged as a key issue likely to prevent widespread clinical adoption of this promising technology. How these issues are being addressed: Cell Guidance Systems' STAR technology directly addresses growth factor cost issues by delivering huge increases in performance of growth factors via simple modifications. Proof of concept for STAR technology has already been demonstrated: In each of the first three growth factors that have now been generated, potency increases in the range 20-200 fold were achieved. Since the manufacturing process is 70% efficient, cost advantages of STAR growth factors are >10-fold. This cost advantage will dramatically reduce reagent costs and allow cord blood and erythrocyte expansion to transition from a lab based technology to commercial reality. Funding Proposal: We are seeking funding for a period of 18 months and aim to generate a series of 10 functional STAR growth factors within a period of 12 months and then use the remaining 6 months to assess the growth factors in CD34+ and erythrocyte expansion systems. It is estimated that each growth factor will cost £10,000-£20,000 to develop