Dendrimeric Peptide-Based Veterinary Vaccines

Protobios LLCkarl@protobios.com

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Contents• Our team

• Current market situation

• Antibiotic resistance

• Peptide-based vaccines as a potential solution

• Our road to market

• Business planning

• Long term roadmap

• Potential obstacles

• Takeaways and highlights

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Our team at Protobios LLC• Karl Märka – Holds a bachelor of science degree from Tallinn

University of Technology in gene technology. Currently doing his Msc degree at Tallinn University of Technology and internship at a biotech startup Protobios LLC specializing in biological data analysis in the field of immunology.

• Anri Kivil – Holds a master’s degree in gene technology from University of Tartu and part of Protobios LLC team specializing in the field of immunology.

• Arno Pihlak – Holds a master’s degree in physics from University of Tartu and works in the field of statistical analysis in Protobios LLC.

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Current situationFive basic facts can generally held to be true in animal husbandry and veterinary medicine:

1. The population of the Earth will continue to expand in the foreseeable future and with it, the need for animal products and the size of livestock herds.

2. The number of antibiotic-resistant bacterial strains and other infectious disease agents will rise in conjunction.

3. Government restrictions on antibiotic use in veterinary medicine are becoming stricter to combat this trend.

4. Vaccination is the single most effective measure to counter the spread of infectious disease.

5. Attenuated vaccine development alone is neither cost-effective nor time-efficient enough to counter the spread of infectious disease in livestock herds.

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What does antibiotic resistance mean?A bacterial strain is said to have become antibiotic resistant if it is able to metabolize one or more types of antibiotic compounds to which it was previously susceptible. This happens through vertical DNA transfer in environments where concentrations of antibiotic compounds are high. Such environments are for example water purification sedimentation pools and the gut of a typical farm cow.

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Current trends in antibiotic resistanceA graph from 2013 study by Marathe et al showing the percentage of bacterial strains resistant to one or more antibiotics from a waste water pool. For consideration, only 11 new antibiotic drugs have been approved by FDA since 1998.

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How does antibiotic resistance affect humans?

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Antimicrobial resistance rates have been on the increase ever since peniccilin was first synthesized and used. The flowchart illustrates the most common route how antibiotic resistant strains are transferred to humans. Adapted from U.S. Animal Health Institute website.

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How are regulatory bodies reacting?

„The European Union (EU), which already forbids the use of antimicrobials to promote growth, plans to strengthen its own rules. Its new antibiotic-resistance strategy, published in November 2011, calls on EU countries to ensure that antibiotics are only available on prescription, and to strengthen surveillance systems to track and report cases of resistance“

Natasha Gilbert, Nature News 2011

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Peptide-based vaccine development as a solutionWith the rising number of antibiotic-resistant bacterial strains, the governments are adopting ever stricter regulations on the use of therapeutical antibiotics in livestock. At the same time, animal epidemics are still causing massive economical losses to livestock owners. One also needs to take into account that antibiotics are only effective against bacteria, which are not the sole cause of disease. Equally important are viruses, fungi and various parasites.

Our goal is to develop cost-effective peptide-based vaccines for use in livestock vaccination. Peptides used in the vaccines are screened and selected bioinformatically using phage display technology. As phage display technology is relatively cheap and fast, new vaccines can be developed when a seasonal outbreak is still in it's infancy or even before that, if blood samples come available. When the relevant immunogenic sequences for use in the vaccine are identified, they can be synthesized in-house or ordered from any of the large number of companies specializing in biomolecule synthesis. Peptides can be covalently attached to a carrier dendrimer, a strong adjuvant can be added and the vaccine sold as a standalone or bundled together with vaccines against other common livestock pathogens.

Main benefit over existing livestock vaccines will be lower development and production costs and the speed with which a new vaccine can be developed and produced in response to an outbreak.

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Peptide-based vaccine development cycle

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Identification of major pathogens, collection and validation of sera.

Identification of pathogen-associated markers with our MVA technology (US patent

14/079,626).

Peptide synthesis and conjugation to

dendrimeric carriers.

Veterinary trials with prototype vaccine.

Marketing and client feedback.

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Proof of concept for the technology

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Protobios MVA technology has been used to identify several pathogen-associated biomarkers. See US patent 14/079,626.

Proof of immunogenicity and adjuvating properties of dendrimers has been established. See HogenEsch, Lu et al 2015.

Proof of immunogenic properties of dendrimeric proteins has been established. See Kumar, Joshi et al 2013.

A pilot study using phage display derived peptide as a vaccine component has been published recently. See Fujiwara et al 2015

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Proof of concept for the technology

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Fuijwara et al 28. February 2015

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Advantages of peptide based vaccines

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• Relatively short and cost-effective development period

• Vaccines are easy to store and handle

• No danger of reactivation in the host

• Antigenic sequences can be varied

• Cheaper to produce

Single dose with 5µg of immunogenic dendrimer costs 0,22€ to synthesize. With additional adjuvant and solution, the manufacturing cost for a single dose would be under 1€. In comparison, conventional vaccines cost from 2,50€ to 27€ per dose.

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Road to market• Most important markets for our product are currently the European Union, United

States and Japan. It is possible to get the necessary certificate to enter all three markets simultaneously through The Veterinary International Committee for Harmonization.

• We have identified enzootic bovine leukosis as the first potential vaccine target. Currently no vaccine or treatment exists for the bovine virus. It is the third most lethal bovine infection worldwide.

• Our first commerical product – an effective veterinary peptide-based vaccine takes, optimistically, about one year to develop since most of the necessary technologies – phage display, dendrimeric carriers etc. have already been developed.

• Clinical trials in veterinary medicine, though much less costly and time consuming than human-oriented clinical trials, should take about 2-3 years to get regulatory approval.

• Once regulatory approval has been granted we can start cooperating with agricultural institutes and veterinary clinics to promote our product. It is our justified belief that our is needed in the current market situation due to increasing pressure by regulatory agencies to curb antibiotic use.

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Product timeline

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Identify market needs in

cooperation with agricultural

experts

Proof of concept for pilot vaccine

Apply for additional investmen

tsProof of efficacy for pilot vaccine

Develop marketing strategy

Customer feedback

Optimize pipeline

Expand company portfolio

Diagnostic kits?

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Market size• In 2010 the total livestock population in the 27 EU

countries amounted to 134 million livestock units (LSU) according to Eurostat

• Livestock population, excluding poultry in the US is over 160 million livestock units according to U.S. Department of Agriculture

• Average lifespan of a farm animal is about 5 years. This means the turnover rate is fast unlike with human vaccinations.

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Veterinary vaccine market

• Current worth is estimated to be 6 billion $

• Estimated to be worth over 8 billion $ by 2018

• Growth rate has been 8% a year

• European market currently has the largest share

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According to: marketsandmarkets.com

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Lifestock losses due to disease

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Average annual lifestock losses due to disease as a percentage of the entire lifestock population. Note that this representslosses in lifestock units (LSU-s). LSU is defined by the mass of the animal. For example, one chicken is 0,015 LSU.The percentages would be much higher when every species are viewed individually. Source: World Livestock DiseaseAtlas.

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Business planning• Expected development costs for the first vaccine are about 50 000€.

Veterinary trials to bring the first complete product to market will cost an additional 50 000€

• Consecutive vaccines will be cheaper as we will already have the necessary certificate for animal studies and the technology will have been optimized. We estimate the average development and trials for each additional vaccine to cost around 80 000€

• Sales are expected to be from 100 000 to 10 000 000 single-shot vaccines per year depending on the targeted pathogen

• Production cost for one single-shot vaccine is estimated to be ~ 1€

• Production will be outsourced to save on costs

• Sales are expected to amount to 10 000 000€ annually in the EU market. This was calculated on the rough assumption of having 4 major commercial livestock species with 5 different major pathogens each and 100 000 single shot vaccines sold annually per pathogen for a price of 5€.

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Long term roadmapSince our end product isn’t really a single vaccine, but an entire pipeline for developing cost-effective, multi-pathogen, peptide-based veterinary vaccines, the possible compounds developed in the long term is limited only by the number of different livestock species and their respective disease causing agents. Due to evolutionary pressure on the pathogens, new strains will eventually rise or cross over from undomesticated species that will also need to be countered.

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Potential obstacles• In pharmaceutical development, the most time-

consuming and costly phase isn’t drug development itself, but rather obtaining valid proof of concept and proof of efficacy through four lengthy phases of clinical trials. The same is generally true in veterinary pharmaceutics, but the regulations are less strict.

• To get through clinical trials a new round of raising capital is often necessary. This is the riskiest step, because funds can fall short and the entire project will have to be shelved or sold. A drug candidate can also fail to show high enough efficacy in the trials and will have to be dropped because of lack of potential commercial sales.

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Highlights and takeaways• Animal husbandry and veterinary science along with it will

continue to be commercially important in the foreseeable future.

• Due to overuse of antimicrobial compounds, many pathogenic strains are becoming resistant.

• Regulatory agencies are pushing new and stricter regulations on antibiotic use.

• Equally important pathogens are viruses, fungi and protozoa.

• Traditional attenuated vaccine development cannot keep pace with the rise of new sporadic and highly infectious pathogenic strains.

• Peptide-based multi-pathogen vaccines developed through translational medicine methods using phage display technology can offer a cost-effective solution.

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