Public Funding for Prokarium Limited

Bladder Cancer (BC) affects more than 550,000 people globally every year. With the highest recurrence rate of any known cancer (80%), it is one of the most expensive for the NHS to treat, costing £65M pa (CRUK). Current immunotherapy treatments based on the weakened strain of _Mycobacterium bovis_ (BCG) present a significant challenge, with substantial impact on patient quality of life and high rates of cancer recurrence. Moreover, chemotherapy and other existing immunotherapies have significant side effects and generally poor response rates. Prokarium's therapeutics are positioned to overcome such limitations by leveraging the natural tumor-suppressive properties of proprietary _Salmonella_ bacteria strain ZH9\. Prokarium have previously shown that ZH9 has superior effectiveness compared to the current standard-of-care BCG in early-stage BC. This has enabled an upcoming first clinical trial and, in collaboration with Switzerland based University of Lausanne and Novigenix, Prokarium aims to expand upon this work to support the further development of ZH9 for BC by exploring the efficacy and mechanism of action of ZH9 in more advanced muscle invasive BC (MIBC), both as a standalone treatment and when administered in combination with other therapies, either already licensed or in development for BC. The incorporation of Novigenix technology will allow understanding of the mechanism of action of the therapy and identification of biomarkers of response that can be used to improve future clinical trials and ultimately provide better treatments to BC patients.

Cancer occurs when cells in the body accumulate genetic mutations that prompt them to grow uncontrollably, affecting normal body functions and potentially leading to death. Acquiring such mutations, and therefore developing a cancer, is more probable as we age. It is estimated that 1 in 2 people will experience cancer in their lifetime. Most standard cancer treatments, such as chemotherapy and radiotherapy, are associated with significant and unpleasant side effects. Therefore, it is a top priority to help patients overcome cancer without having to rely on toxic drugs that severely affect their quality of life. The use of microbe-based immunotherapy for cancer treatment was first described in the late 19th century, and the current standard of care for bladder cancer, the bacterial therapy using the Bacillus Calmette-Guerin (BCG), was medically established in the 1920s. However, a lack of molecular tools and the increasing popularity of radiotherapy in a pre-penicillin world stalled further microbial immunotherapy research. Now, with modern synthetic and molecular biology tools and technologies, the approach of using bacteria to train a patient's own immune system to recognise and fight cancer cells has re-emerged. Using cutting-edge bioengineering technologies, Prokarium has developed a treatment platform based on safe, attenuated Salmonella bacteria capable of navigating towards and colonising the tumour environment, with a promising first product reaching bladder cancer patients in late 2023\. Through an ongoing partnership with the London Biofoundry, Prokarium have set up a pipeline that can load Salmonella with modular protein blocks that can be delivered to tumours and expressed by tumour-associated cells to activate or suppress different elements of the immune system. In this project, Prokarium aims to combine some of the DNA technology and bacterial strains that have already been developed with the natural tendency of Salmonella to enter human cells to engineer a new Trojan horse drug delivery platform that is able to attack cancer cells from within. This approach is designed to either supply new drugs that are released from within the cancer cells (eliminating them in the process) or establish a long-lived depot of drugs that can be released in cycles to maintain long-term therapeutic activity. Ultimately, this work is designed to provide cancer patients with readily available and much needed new therapy options.

Enteric fever is caused by two types of bacteria: Salmonella Typhi (typhoid) and Salmonella Paratyphi (paratyphoid), contracted from contaminated food or water. Although there are vaccines to prevent typhoid, there is no vaccine for paratyphoid which is a growing problem globally. With nearly 27 million global cases of enteric fever each year, Entervax™, the dual typhoid-paratyphoid vaccine developed in this project, could profit not only the Mexican society but also populations in developing countries and travellers globally. This project brought together UK-based Prokarium’s novel vaccine delivery technology with Mexican Probiomed’s pharmaceutical manufacturing capability. The Entervax™ vaccine is swallowed, passes through the stomach and into the intestine where safe bacteria are released to target the body’s own immune cells and trigger an immune response to protect against enteric fever. This avoids the need for injections, and more importantly means it can be delivered to people living in remote areas, as the vaccine is self-administered. If the future vaccine is manufactured for market supply in Mexico, this would generate and support many highly skilled jobs in the pharmaceutical industry.

As recent disease outbreaks show, the world needs to be faster and better at developing, manufacturing, testing and distributing vaccines. Prokarium, a UK-based vaccine development company focusing on oral vaccines, can move from bench to clinic 6-12 months faster compared to most injectable vaccines, use the same manufacturing process to produce a wide range of vaccines, cut costs by up to 70% and clinically test its oral vaccines under very simple out-patient conditions. Prokarium’s delivery strain has established a strong safety record in 10 phase 1 and 2 clinical trials in 471 volunteers including 101 children. In this project Prokarium will clinically trial its next generation vaccine platform ‘Vaxonella’, which when commercialised can be distributed at temperatures of up to 40 C for at least 12 weeks and which in emergencies can be self-administered without the need for medical personnel. These are the vaccine characteristics needed to combat serious diseases and potential epidemics such as plague caused by the bacterium Yersinia pestis. Historically plague was responsible for several epidemics, including the Black Death, which killed over 50 million people in Europe in the late medieval period. Today plague still kills people and in recent years the number of countries where plague is endemic increased, resulting in the World Health Organization labelling it a re-emerging disease. Coupled with the risk of future antibiotic resistant strains of Y. pestis arising, and its potential as an aerosol-delivered bioterrorism weapon, there is a need to develop an effective vaccine. An ideal plague vaccine must be designed for use in low- and medium-income countries to immunise high risk populations (e.g. Madagascar, DRC, etc.) on a seasonal basis and to rapidly distribute in response to an outbreak caused by major disturbances such as floods or earthquakes when the reservoir populations are disturbed. Prokarium’s Vaxonella platform is based on live strains of Salmonella bacteria which are weakened so that they cannot cause disease. The Salmonella are taken orally as a capsule or liquid resuspension for children, they transit the stomach into the small intestine where they actively enter through the gut lining into the antigen-presenting cells of the immune system. The Salmonella to be used in this project have been programmed to express two protein antigens from Y. pestis from within the body’s own immune cells. This targeted vaccination means all arms of immunity are triggered including the first line of defence, namely mucosal immunity. Also, manufacturing is up to 70% cheaper because costly purification needed for injectables is eliminated. Prokarium has already successfully tested a plague vaccine in mice. Now it will confirm that an improved vaccine can protect mice against Y. pestis, then rapidly manufacture a liquid suspension, and then test it on human volunteers in a clinical trial, where the safety and immune response will be measured. This will provide information to support further development of the plague vaccine and crucially be the first proof of concept of the next generation Vaxonella platform that could transform the way vaccines are developed, manufactured and distributed.

In February 2016 the WHO declared the Zika virus (ZIKV) to be a “Public Health Emergency of International Concern”. Experts now believe ZIKV is linked to a broad set of complications in pregnancy, including miscarriage, stillbirth, premature birth and eye problems with 29% of scans showing abnormalities in babies in the womb, including growth restrictions and microcephaly, in women infected with ZIKV. To clear viruses, the body generally requires both systemic and cellular immune responses, but unlike most insect-vectored diseases, ZIKV can also persist in semen and has been shown to spread as a sexually transmitted disease. That means mucosal transmission by travellers returning from any of the 43 endemic countries (population 525 million; 66 million tourists p.a.) may bring back and spread the disease in non-endemic countries. ZIKV has also been found in other bodily fluids including saliva and urine. Therefore, a vaccine that triggers systemic, cellular and mucosal immunity is needed, and preferably one that is easily manufactured and distributed under many different conditions. Prokarium’s oral vaccine platform, Vaxonella, mimics an intracellular pathogen and expresses vaccine antigens from within the immune cells of the gut lining. This means Vaxonella triggers all arms of immunity while reducing manufacturing costs by up to 70% due to the elimination of downstream purification of protein, which is always needed for injectable protein vaccines. In addition, Prokarium has developed a dried formulation of Vaxonella that is thermostable at 40 C for up to 12 weeks. These qualities are specifically suited to ZIKV and distribution to endemic areas as well as travellers, as Vaxonella vaccines can be produced almost anywhere and be self-administered. This temperature stability and self-administration will be useful in remote areas lacking medical personnel, but also for travellers if a booster is needed, as most travellers do not prepare ahead of time to take several doses of vaccine. Vaxonella is based on live strains of Salmonella bacteria that are weakened so that they cannot cause disease. The Salmonella are taken orally as a capsule (or as a liquid suspension for children); they transit the stomach into the small intestine where they actively enter through the gut lining into the antigen-presenting cells of the immune system (mimicking an intracellular pathogen). Using Prokarium’s proprietary genetic technologies, the Salmonella to be used in this project will be programmed to express protein antigens from ZIKV, and tested in mice to study the level of immune response generated. The best candidates will then be used to immunise mice which will be challenged with the actual ZIKV, using the existing subcutaneous challenge model at Public Health England (to mimick the mosquito bite route), who will also develop a novel vaginal challenge model (mimicking the sexually transmitted route), to see if they are protected compared to unvaccinated control mice. This will provide information to support further development of the ZIKV vaccine for future evaluation in human volunteers and represents the first known attempt to make a ZIKV vaccine that has a high chance of preventing both the insect-vectored and sexually transmitted versions of the disease.

C. difficile infection (CDI) causes severe diarrhoea in hospital patients after treatment with broad-spectrumantibiotics. CDI can be successfully treated with specific antibiotics, but infection and diarrhoea re-occurs in upto 3 out of 10 patients and on average 2 of these will die. A vaccine could prevent CDI but none is currentlyavailable. The vaccines currently in clinical trials use inactive C. difficile toxins to generate an immune responsebut not all patients respond. Two UK companies, Absynth and Prokarium, are collaborating to create the firstoral vaccine against CDI. The vaccine is based on combining a safe living bacterium already tested in clinicaltrials, with novel vaccine antigens that offer a non-toxin based approach with potentially broader protectionagainst disease. The vaccine delivered to the site of infection, will target the colonising bacteria, so infectioncould be prevented and those vaccinated are unlikely to become asymptomatic carriers. After this Innovate UKsupported project, the companies hope to gain additional investment to progress the vaccine into clinicaltrials

New vaccines are made from proteins that have to be injected using a needle. The process of making these proteins is very expensive and different for each vaccine. They cannot be put into tablets because they would be digested in the gut, and they need to be refrigerated and transported around developing countries via a distribution network called the cold chain. This project aims to create a way of making vaccines which is cheaper, requires only one manufacturing process regardless of the type of vaccine, and allows the vaccine to be swallowed as a capsule. These capsules will be designed to be stable without refrigeration for the few critical weeks needed for distribution between cities and remote villages, thus breaking the final link of the cold chain and allowing people to vaccinate themselves. We use a safe strain of Salmonella in the capsule, which produces the required vaccine inside the human body. The technology may revolutionise the way all protein vaccines are delivered and the first proof will be in the form of a vaccine against typhoid and bacterial diarrhoea, which cause hundreds of thousands of deaths annually.

Prokarium Ltd of Keele, Staffordshire, has invented Vaxonella, a synthetic biology platform that promises to eliminate the need for needles for many vaccines. This is not only convenient for UK travellers going abroad, but will be essential in preventing diseases in rural areas where people do not have access to medical professionals. Vaxonella is based on a safe bacterium that has been taken orally by hundreds of volunteers in clinical trials in three different countries. Prokarium’s technologies allow it to programme this bacterium to produce almost any kind of protein vaccine from within the gut’s own immune cells. The financial help from the UK government’s Technology Strategy Board and collaboration with the University of Birmingham will allow Prokarium to test Vaxonella for preventing diarrhoea caused by Enterotoxigenic E. coli, Clostridium difficile and Typhoid, which infect millions and kills hundreds of thousands of persons annually, many of them children and the elderly.