ProxAgen Ltd. is a biotech company devoted to the development of prototypes for human and animal vaccines and bioassays. Company’s main purpose is to work as interface between Research Institutes, and Industries producing bioassays and vaccines, for facilitating the development of new products based on innovative platforms, combining basic research, applied technologies as well as all the pre-clinical and clinical studies necessarily for the validation of the developed products, and the exploitation of the industrial performance.
ProxAgen General Activities in a nutshell
Basic research, applied technologies for the development of new diagnostic assay and vaccine prototypes. Large scale (genomic) T- and B-cell antigens and epitopes identification throughout immunobioinformatic analysis. Pre-clinical and clinical studies necessarily for the validation of the developed products. Promote excellence and technological development in the biomedical field, specifically in the fields of diagnosis and vaccines for medicine and veterinary. Carry out feasibility studies, surveys, development plans, and design interventions in production facilities to launched production activities in the field of its interests;
Plan, organize and manage theoretical and practical education and training.
ProxAgen Ltd has been founded in July 2009 in Bulgaria. Researchers of the University of Rome “Tor Vergata” together with another Bulgarian shareholder gave live to a pivotal instrument to conduct research and development in the field of vaccine and diagnostic against infectious diseases. The idea of creating ProxAgen, however, born long before from the long term collaboration among the University of Rome “Tor Vergata” (Italy), the BulBio-NCIPD and the National Center of Infectious and Parasitic Diseases of Sofia(Bulgaria) in the field of vaccine development. In fact, a number of agreements for collaborating in the development of different vaccines were established among the Institutions.
These are here summarised:
1. the partnership agreement for the development of a vaccine co-formulation between the anti-tuberculous vaccine M. bovis BCG and HIV-peptides in single administration among the BulBio-NCIPD Ltd. (Sofia, Bulgaria), the National Center for Infectious and Parasitic Diseases (Sofia Bulgaria) and the University of Rome “Tor Vergata” (Rome Italy), signed on April 6th 2006;
2. the subsequent operative Memorandum of Understanding “Regarding the research agreement for the development of a paediatric HIV vaccine based on a combination of BCG plus HIV-1 specific peptides”, among: BulBio-NCIPD Ltd (Sofia, Bulgaria), REsearch for Drug Development - RE.D.D Srl (Spin-off of the University of Rome “Tor Vergata”; Rome, Italy), and S.A.R.M. Srl (Rome, Italy), signed on January 9th 2007;
3. the general agreement for the “Development of a new co-formulate anti-tuberculous and anti-Hepatitis B vaccine, including pre-clinical and phase I and II clinical studies”, among: BulBio-NCIPD Ltd (Sofia, Bulgaria), HEBER BIOTEC S.A, (International representative of the “Center for Genetic Engineering and Biotechnology” - Havana, Cuba), the National Center of Infectious and Parasitic Diseases (Sofia, Bulgaria), the Università di Rome “Tor Vergata” (Rome, Italy) and the Technology Development for Africa Srl (Spin-off of the University of Rome “Tor Vergata” - Rome, Italy), signed on October 20th 2008.
4. the involvement of the different Institutes and Researchers of the University of Rome “Tor Vergata” (Rome, Italy) as active partner of the “Latium Vaccine Pole”: a consortium of 11 private and public Research Institute of the area of Rome for vaccine development.
As matter of fact, to quickly address the specific needs of rapid developing the collaborating activities ProxAgen was put on the way to be founded.
ProxAgen acquired and developed a number of technical platforms throughout its Shareholders for the development of vaccine prototypes and innovative diagnostics. These include:
Immunobioinformatic facilities for T and B cell epitope selection on genome scale.
An important aspect in the development of diagnostic system and/or vaccine, is the rapid identification of relevant antigens to which virtually all the subjects could respond ordevelop a protective immune response.
A strong limitation to the identification of antigen is represented by the immunogenetic restriction of the T-cell response as well as by the difficulties in identifying the area of antigens that are soluble exposed to be recognised by antibodies.
The recent identification of the typical pattern of recognition of microbial antigens by T- and B-cells are at the heart of the bioinformatics approaches applied to immunolgy.
These approach that is known under the name of “reverse immunogenetic” approach has been successful to define T-cell epitopes and to find out new peptide epitopes that can be used to assess T-cell response. Similarly, molecular modeling has been used to identify portion of protein that could be more probably exposed on the water accessible surface and thus can be target of the B-cell recognition through antibody.
Researchers of the University of Rome “Tor Vergata” partecipating directly in ProxAgen, have developed in the recent years experties and methodologies in the field for the reverse immunology. Specifically, bioinformatic tools for the identification, enumeration and characterization of peptide binding epitopes to HLA molecules in whole genomes have been developed together with system for molecular modeling of proteins by ab-initio and homology methods.
The immunoinformatic platform for T-cell epitope identification allow the crossing of whole genome protein sequences on HLA-peptide binding motif databases using an algorithm of quantitative implemented peptide biding motif analysis in order to identify high immunogenic proteins targeting T-cell immunity. The platform enables the characterization of the immunogenicity of whole genomes, the identification in the genome of the (I) most immunogenic targets, (II) T-cell epitopes and HLA-promiscuous multi-epitopic peptides in each single protein, (III) the enumeration of epitopes recognized in the genome by single or by a couple of HLA-alleles miming the recognition at single subject level. Further, the same platform is equiped with algorithm for secondary analysis of protein for indentify potential area of targeting B-cell response.
Other Immunobioinformatic facilities
For the identification of B-cell response targets, the molecular modeling analysis conduct both by ab-initio and homology system allow the development of three-dimentional models of the target protein. The refined model could be analysed for the solvent exposed portions for the identification of potential conformational B-cell epitopes.
These represent part of the bioinformatic platforms for a comprehensive analysis of antigenic sequences available at ProxAgen, thereby allowing the rapid identification of useful targets within a genome for the development of T- and B-cell diagnostics and based vaccines.
BCG adjuvancy for proteins and peptides
Due to the epidemiology of tuberculosis, many countries in Africa, Asia, America and also in Europe are routinely use the BCG vaccination within the first week of the childbirth. Thousand of BCG vaccinations performed in children infected with HIV at the birth indicate that this kind of vaccination is safe also in immunodepressed children and dissemination of BCG is a rare event. Further, literature shows no evidence for negative effects of BCG on the response to other vaccines even in HIV+ children, and the vaccine is considered safe irrespective of the mother’s serostatus.
Recent publications examined the direct interactions of BCG and conventional vaccines in healthy children. In these studies, BCG had a significant positive effect (an adjuvant effect) on the response to most of the vaccine antigens that were administered at the same time. This finding is critical to our vaccine platform concept, because it suggests that antigens co-delivered with the mandatory BCG immunization will benefit from a strong adjuvant effect of the mycobacterium.
BCG has been shown to possess a strong systemic and mucosal adjuvant activity, which can induce both humoral and cell-mediated immune response. In particular, viable BCG has been described inducing the signalling via both Toll-like receptors (TLR2 and TLR4) and causing the maturation of immature DC into fully activated mature DC. Moreover, infection of DC with BCG facilitates secretion of pro-inflammatory cytokines (IL-1, IL-12, TNF, IFN, etc.) and up regulates CD40, CD80, CD83, CD86, and MHC class I molecules. Moreover, DC exhibit potent antigen presenting ability through uptake of BCG, and this activity can be used on soluble antigens other than BCG itself. Finally, conditioned DC can be a temporal bridge between CD4 TG helper and CD8 cytotoxic T cells.
Another class of T lymphocytes, which has been described to be activated by mycobacterial products such as lipid and glycolipis, are the CD1-restricted T cells. Glycolipid antigens derived from M. tuberculosis stimulate CD1-restricted T lymphocytes, and cells reactive to glycolipid have been found in patients with tuberculosis. The CD1-restricted T lymphocytes as well as the NKT population, which represents a subtype of these cells, play an important role in the activation of immune response, since are able to produce high levels of both IL-4 and IFNg upon activation..
Furthermore, recent studies on the influence of BCG on antibody and cytokine responses to human neonatal vaccination have shown that BCG induces a potent Th1 response both to mycobacterial and to unrelated antigens. In fact, administration of BCG at the time of priming (at birth) markedly increases the cellular and antibody responses to the hepatitis B vaccination, and a limited effect on the tetanus and diphteria toxoids which are administered at two months of age.
Finally, BCG has been also described to induce a potent B cell response with production of IgG and IgA, also against unrelated vaccine antigens administered mucosally, as shown by the increased antibody response to oral polio vaccine.
Therefore, in order to meet the immediate threat of infectious diseases, we selected a platform strategy that seems likely to encounter fewer regulatory obstacles, largely due to the fact that we are not modifying, or only minimally, any existing products and we simply administer a second vaccine product at the same time as BCG in a co-formulated way. This will be evaluated, and the platform implemented, since the first products to develop by the co-formulation of BCG with Hepatitis B protein as well as BCG with HIV peptides.
T-cell assays for diagnosis and monitoring infectious diseases and monitoring vaccine protocols
Beside the identification of relevant antigens, another limitation to developing efficient system for the immunodiagnosis and monitoring the T-cell response against infectious diseases, as well as for monitoring vaccine protocols, is represented by the use of reliable techniques. In fact, from one side these techniques should be easily automated and routinezed by using analytical instruments already available in most clinical laboratories, such as flow-cytometric analysis and ELISpot. On the other hand, the techniques should be also robust and easy to perfome in different clinical setting, including work on field in third countries.
Researchers of the National Center for Infectious and Parasitic Disease and of the University of Rome “Tor Vergata” partecipating directly in ProxAgen have developed specific expertises in T-cell monitoring throught flow cytometry and ELISpot assays. A specific unit will be established in ProxAgen to further develop these technologies to define innovative parameters to be specifically used for measuring T-cell response and be easely applied in different clinical settings.
Small Biomanufacturing and GMP production
One of the critical, both economical and technical, aspect in developing vaccine is to have easily availability of small batches of Good Manufacturing Preparation (GMP) products for phase I and II studies. In general, companies that choose to build their own manufacturing capacity often do so to optimize long-term control and economics. However, it typically costs tens if not hundreds of millions of dollars and takes 3-5 years to build, equip and validate a traditional biomanufacturing plant. This means significant capital must be committed during the riskier early stages of a molecule’s development.
Alternatively, biotechs can look for partner with production capability both for preliminary studies and, ultimately, commercial production.
Furthermore, also the size of preparation is critical and cost affecting the vaccine product development. In fact, usually only small medium production are necessarely for vaccine development purpose. For large companies performing GMP this scale is usually critical and determine overcosts for the time to be dedicated for the production line for specific GMP purpose.
To address these challenges, the most valid alternative is represented by bioprocessing manufacturing platform built almost exclusively with disposables technology. Xcellerex Ltd (Boston, US) has developed a suite of bioprocessing manufacturing platform built almost exclusively with disposables technology, overall named “FlexFactory.” The FlexFactory is organized into several discrete modules, each of which is self-contained in its own controlled environment. The FlexFactory manufacturing strategy delays the biotech’s facility build-out for as long as possible and greatly reduces investment risk (overall investment is significantly reduced too due to the simplified facility requirements). In addition, because of its modular design and the disposable platform, the FlexFactory can easily be adapted for multi-product manufacturing.
ProxAgen is going to acquire and establish as platform within the first three years of activity the FlexFactory for its own production as well as to offer the small biomanufacturing and GMP production to third parties. This technology would strongly increase the portfolio of the Company and the possibility of collaborations with new products development.
Platforms available in out sourcing
A part the specific platforms in house available at ProxAgen, other technological platforms in the fields of vaccine research and diagnostic development, are directly available to ProxAgen upon specific collaboration through the NCIPD, BulBio-NCIPD, the University of Rome “Tor Vergata” and the Latium Vaccine Pole.
In fact, nowadays, only a multidisciplinary and integrate approach to vaccine development is required to overcome the previous vaccine models of selecting single antigens based on availability, immunological data and personal preference of the scientist.
Further, the availability of Animal Stations (University of Tor Vergata and Istituto Zooprofilattico Sperimentle Lazio e Toscana) for preclinical investigations would rapidly allow the efficacy of vaccines, which could be tested alone, with and without adjuvants, and in prime-boost strategies in an iterative process optimising the next experiment based on data on protection and immune response from the previous experiment.
Finally, the close interaction between research groups and health centres (National Institute for Infectious Diseases “L. Spallanzani”, Policlinico Gemelli e Policlinico Tor Vergata) would also allow patients recruitment for Phase I and II clinical trials, when appropriate.
The other technological platforms available in outsourcing is including:
1. Phage display identification of target vaccine
2. Identification of antigens in host-pathogen interaction models
3. Innate immunity and T-cell activation systems
4. Adenovirus delivery system
5. Antigen Production in plant systems and virus like particles
6. Lyposomes delivery systems
Policy in the specific area of intervention of ProxAgen
Vaccines are the most effective public health measures developed and have saved million of lives. Vaccination programs represent one of the most cost efficient health intervention developed, and has resulted in smallpox being eradicated and many diseases being under control like polio, hepatitis A and B, tetanus, diphtheria, meningitis due to Haemophilus influenza and Meningitis A, C, Y, W135, measles, mumps and rubella and pneumococcal infections. Lately, vaccines against human papilloma virus have shown great promise to reduce cervical cancer.
Vaccines have been developed against a variety of bacteria and virus, but chronic, life-long diseases like hepatitis, AIDS, tuberculosis, malaria, toxoplasmosis and leishmaniasis have been difficult to prevent despite a huge research effort over the past two decades. Furthermore, the possibility of using vaccines not only for prevention but also for immunotherapy of individuals already infected is a goal given that drugs cannot eradicate many of the chronic infections. For instance, toxoplasmosis, hepatitis B and C, HIV and also tuberculosis are chronic infections where the disease can remain asymptomatic or latent for years and current therapies are ineffective and with many side effects. For these infections an immunization after infection, or a vaccine which work an immune therapy, is needed.
Vaccine development is a long and difficult process where the selection of antigens is often disorganized based on different studies for immunogenicity assesment, analysis of humoral or cellular immune response, animal study and effectiveness of the vaccine products. Also, the choice of antigen to be studied is often not completely rational, and many antigens are not tested, and most protein-encoding open reading frames (ORFs) predicted from sequencing projects have remained completely uncharacterized at the functional level. Therefore, many antigens involved in protective immunity will be missed if not studied in a systematic way.
Industry for vaccine and diagnostic fopr infectious diseases worldwide
While recent technologies have expanded the horizons for new and improved vaccines and diagnostic test for infectious diseases, considerable financial and staff resources must be available to support the full development of these proucts. From the time that an initial lead has been identified, it can take 10 years and well over $100 million to develop a new vaccine. Ten to 100-hundred fold less is necessarily as investment for the development of a new diagnostic test.
Despite these limitations, the number of companies developing and producing vaccines and diagnostic tests, and in particular small medium enterprises that are devoted to specific vaccine and diagnostic products development, or just a part of the process, has been considerably expanded in the past decades, in particular in US.
With the time, it becomes increasingly uncommon that any single organization has all the technologies for the design, development, production and commercialization of a vaccine and/or a diagnostic test. Therefore, the different steps of development and production, including the in-licensing and business development, have been areas of increasing activity from separate entities. In fact, most of the licensors are represented by academic and/or government laboratory or a small biotech company.
With the time not only the antigen but also the technology for vector, method for discovery or screening of antigens, production method, formulation, device, or combination of such inventions have became part of the licensing for which the necessary financial, technical and physical resources can be available only by agreements with production entities.
From an economical point of view, the vaccine and diagnostic market was considered once "very small and fraught with the inability to price effectively". On the contrary now, with fewer large manufacturers hawking new products, the business is expected to grow rapidly the next few years in all the pipeline. About this, recently analysts at Frost & Sullivan say the booming this market will more than double in the next coming years leading up to 2014. The report says that novel vaccines and innovative technology pushed industry revenue to $4.5 billion in 2007 and it is expected that figure to jump to $9.85 billion in 2014, making the vaccine market a potential blockbuster. In general, vaccine and diagnostic manufacturers have responded to the growing market by investing much more into research and development. Despite this, it is critical to note that the European market has dragged behind the US.
Strategy for industrial development
Although annual deaths and lost years of healthy life from infectious diseases have decreased over the past decade, the worldwide impact from infectious diseases remains substantial. Overall, infectious diseases remain the second leading cause of death worldwide.
Of the estimated 57 million deaths occurring worldwide annually, about 15 million, >25%, are directly caused by infectious diseases. Millions more deaths are due to secondary effects of infections.
In the United States, the Centers for Disease Control and Prevention has devised strategies to prevent, monitor, and contain disease outbreaks. Within the NIH, the National Institute of Allergy and Infectious Diseases (NIAID) is the lead agency for infectious disease research. Over the 10 years, the NIAID budget has quadrupled; spending on emerging infectious diseases has increased from <$50 million in 1994 to >$1.7 billion projected for 2005.
In the last decade, many International Foundations have provided a strong impetus for vaccine and diagnostics research and development. For instance, the Gates Foundation’s commitment to the Grand Challenges in Global Health, which was launched in 2003 to accelerate the discovery of new technologies to improve global health, has committed over $450 million to support more than 40 projects on topics such as making childhood vaccines and diagnostic easier to use in poor countries.
With regards to the private sector, it is now evident that vaccine and diagnostic development is a growing business area.
Therefore, with this overall picture, large cost and high risk, it is becoming more important for the design of new vaccine products and their implement to produce a Product Development Plan very early during the time-period of the development in order to map out all the technologies and resources (money, people, facilities) necessary for optimizing the likelihood of success of the program.
Need of a new company
It is clear, from the above mentioned analysis, that with the market expansion in the vaccine and biotech areas that is characterizing this period, different expertises are nowadays necessarily for developing a new vaccine or a diagnostic product. Only an entity bridging together and coordinating activities among different structures interested to the vaccine development (such as Academic, Research Institutes and private bodies), would provide the necessarily flexibility to carry out quickly the most critical parts or the complete chain of research and development for the vaccine and diagnostic products.
ProxAgen, taking light from the long term collaboration among Bulgarian and Italian research institutions, with the flexibility of a spin-off and developing specific platforms and expertises, represents the right instrument to facilitate the development of new vaccine and diagnostic products.