Novi pristopi za boljša biološka zdravila (Slovene)

Biological drugs are increasingly implemented in modern medicine as an alternative to conventional synthetic drugs. The concept of biological drugs describes all medicines derived from biological sources (for example, also blood and blood products). However, in a stricter sense, this term applies especially to recombinant therapeutic proteins. Perhaps the most well known example are monoclonal antibodies based on the immunoglobulin G1 (IgG1), which have in the recent decade shown great success in the treatment of cancer. The main advantage of using monoclonal antibodies compared to using classical cytostatics is in their ability to specifically bind to target proteins that are expressed to a greater extent in cancerous cells but less so in non-cancerous. Triggering of antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) is often desired in therapeutic antibodies. In some cases, however, it is better that these immune functions are not activated. A typical case, is ipilimumab, an IgG1 monoclonal antibody used primarily in the treatment of the melanoma, a type skin cancer. Ipilimumab activates the immune system by binding to the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expressed in dendritic cells and causes the activation of cytotoxic T-lymphocytes. This binding to CTLA-4 blocks the inhibitory signal and enables T-lymphocytes their natural cytotoxic activity against cancer cells. ADCC and CDC, which are associated with the ipilimumab's Fc region are not required for ipilimumab to function – on the contrary – they are a known cause of its side-effects. One of the more serious is the inflammation of the pituitary gland (hypophysitis), caused by ipilimumab's Fc region binding to the complement 1q and also to Fcγ receptors. The aim of this research is to prevent the binding of ipilimumab to Fcγ receptors and complement 1q, which are the cause for its unwanted side-effects, using a novel computational approach. Our approach will differ from other state-of-the-art methods for introduction of point mutations (or their combinations), in the fact, that we will replace whole substructural elements of the protein structure (for example, entire loops). These elements will be obtained from existing human proteins that have been proven stable by evolution and will presumably not trigger immune response. The obtained mutants will be experimentally tested in vitro and in vivo. The aim of the proposed research is also a novel computational approach for the modulation of therapeutic protein function. In this approach we will further develop and extend our ProBiS algorithm and ProBiS web severs for prediction of protein binding sites and protein ligands, drug repositioning and prediction of off-target effects, which are already well accepted in the scientific community and widely used by many research groups world-wide for drug development. Our new approach will allow the replacement of a section of acceptor protein structure with a similar section of donor structure using ProBiS's ability to detect locally similar patterns of physico-chemical properties on protein surfaces. Donors will be chosen from the pool of human proteins from the Protein Data Bank. The therapeutic protein - acceptor, will therefore obtain the desired properties of its donor, for example, it will be Fc silent due to its reduced affinity to Fcγ receptors, it will be stable, and it will not be immunogenic due to the use of existing substructural elements that our immune systems already know. The computational approach will be developed, tested and used, for the first time, on the therapeutic protein – ipilimumab, a humanized monoclonal antibody used primarily for the treatment of melanoma. This new approach will be freely available to a broad research community and will enable the design of proteins with new desired properties.

We will develop an original approach to design therapeutic proteins with improved properties. Originality of our approach will be in the new way of identifying elements of protein structures suitable to be used in new environments with new functions. Original will be the use of the new approach on the CH2 subunit of the Fc region of IgG1 to silence the interactions with Fc? receptors, which has not been investigated yet. The developed approach will be validated and used for the design of novel monoclonal antibodies that should exhibit fewer side effects in the treatment of cancer. The result of this project will be improved biological drugs for the treatment of cancer with fewer side effects. The cooperation with Lek d.d., will allow for not only a scientific study of high interest, but also the potential for commercialization of our results. The result will be freely accessible and generally useful computational tools to design mutants of therapeutic proteins. The tools will be useful with various proteins, not only with the IgG antibodies. We expect patents and publications in top scientific journals. By combining new computational methods with experiments on a very relevant issue – therapy of cancer – which will ensure high relevance of our study.

Biosimilars and improved biological drugs (biobetters) are the fastest growing segment in the pharmaceutical industry; in 2013 the global market for biosimilars was USD 19 billion and is expected to be worth USD 100 billion by 2020. Therefore we are witnessing an accelerated investment in biosimilars and improved biological drugs. Biological drugs are also a great opportunity for Slovenia. Slovenia currently holds, together with Sandoz, a pioneering position and is ahead of many developed countries. We believe that the development of biosimilars that began in collaboration between Lek d.d. and National Institute of Chemistry, is an example of a success story on which it pays to build on. Today, Lek's biopharmaceutical unit located in Mengeš is one of the four main biopharmaceutical development and production centres within Sandoz Biopharmaceuticals. Biopharmaceuticals Mengeš is a cell line development centre of excellence for biosimilars, centre of excellence for PEGylation for Novartis group and process development centre of excellence for cell culture derived biopharmaceuticals. It is the only industrial centre for recombinant biotechnology in Slovenia. In Mengeš, there is also a global center of excellence for bioinformatics. Head of the proposed project has collaborated with Lek Biopharmaceuticals for several years on modeling and design of biological drugs. In the last two years, he led and successfully finished three industrial projects with Lek Biopharmaceuticals. Using the ProBiS approach for local structure alignment of proteins and prediction of protein binding sites and CHARMM program package for molecular simulations, also co-developed in the department, he has already designed mutants of biological drugs with improved properties and simulated the behavior of biological drugs in different experimental conditions. These projects resulted in promising initial experimental results. Globally in the biologics segment it is evident that the area of computer modeling and simulation in the design process of biologicals is relatively underdeveloped. This is partly due to the great complexity of the process which requires combined knowledge of molecular biology, genetic engineering, system biology, analytics, computer modelling, etc. In Slovenia, individual expertise have been developed to the highest level. Their integration represents a niche opportunity for Slovenia and will enable innovative development of improved biological drugs. In view of the fact that the value of the development of an improved biological drug may amount to several billion USD, the proposed novel computational approaches represent an opportunity for large savings.