Univerza na Primorskem Fakulteta za matematiko, naravoslovje in informacijske tehnologije
Več informacij o projektu / More info about the project
Vsebina projekta / Project content
Biološka zdravila se vedno bolj uveljavljajo v medicini kot nadomestek klasičnih sinteznih
zdravil. Pod pojmom biološka zdravila razumemo vsa zdravila pridobljena iz bioloških virov
(na primer tudi kri in krvne izdelke), v ožjem smislu pa predvsem rekombinantne terapevtske
proteine. Najbolj znan primer so monoklonska protitelesa na osnovi imunoglobulina G1
(IgG1), ki se zadnje desetletje vedno več in vedno bolj uspešno uporabljajo tudi za
zdravljenje raka. Prednost uporabe monoklonskih protiteles pred klasičnimi citostatiki je v
njihovi specifični vezavi na tarčne proteine, ki jih rakave celice izražajo v večji meri kot
nerakave.
Aktivacija s protitelesi povzročene celično posredovane citotoksičnosti (angl. »antibody
dependent cell mediated cytotoxicity”; ADCC) in s komplementom povzročene citotoksičnosti
(angl. »complement dependent cytotoxicity«; CDC) je pogosto zaželena lastnost
terapevtskih protiteles, vendar obstajajo primeri protiteles, pri katerih je zaželeno, da do
aktivacije te imunske funkcije ne pride. Tipičen primer zdravila, pri katerem udeležba ADCC in
CDC ni zaželena, je biološko zdravilo ipilimumab. Ipilimumab je monoklonsko protitelo IgG1,
ki se uporablja za zdravljenje melanoma, vrste kožnega raka. Ipilimumab aktivira imunski
sistem – citotoksične T limfocite, tako da se veže na protein citotoksični T-limfocitni antigen-4
in prek te vezave izklopi zaviralni mehanizem, kar omogoči T-limfocitom citotoksično
delovanje na rakave celice. Ipilimumab prek svoje Fab regije torej deluje kot aktivator
citotoksičnih T-limfocitov, ki nato uničijo rakave celice. Udeležba ADCC in CDC, posredovana z
Fc regijo, pri tem ni potrebna za funkcijo, in je znan povzročitelj stranskih učinkov tega
zdravila. Eden hujših je vnetje hipofize (hipofizitis). Ugotovili so, da je za nastanek
hipofizitisa kriva vezava ipilimumaba na del komplementa 1q in levkocitne Fcγ receptorje,
zaradi česar pride do aktivacije omenjene vnetne kaskade.
Cilj raziskave bo preprečiti vezavo ipilimumaba na Fcγ receptorje in del komplementa 1q, ki
povzročata neželeno citotoksičnost tega biološkega zdravila, z novim računalniškim
pristopom. Naš pristop se bo razlikoval od do sedaj uporabljanih metod, na primer uvedbe
točkovnih mutacij posameznih aminokislin v tem, da bomo zamenjevali večje elemente
proteinske strukture dolžine od nekaj do okoli 10 aminokislin, na primer zanke, ki že
obstajajo in so skozi evolucijo preizkušeno stabilne v drugih proteinih. Prednost naše
metode bo, da bodo načrtovane mutante pridobile nove lastnosti, hkrati pa bodo ohranile
stabilnost in ne bodo imunogene. Načrtovane mutante bomo nato eksperimentalno
ovrednotili in vitro ter in vivo.
Cilj raziskave bo tudi nov računalniški pristop za modulacijo funkcije terapevtskih proteinov.
Novi pristop bo nadgradnja ProBiS algoritma in ProBiS spletnih strežnikov za napovedovanje
vezavnih mest in ligandov proteinov, odkrivanje novih zdravilnih učinkovin in napovedovanje
stranskih učinkov zdravil. ProBiS je dobro sprejet v znanstveni skupnosti in je široko
uporabljan v raziskovalnih skupinah, ki se ukvarjajo z razvojem novih zdravilnih učinkovin.
Naš novi pristop bo omogočal presajanje delov strukture proteina – akceptorja s podobnimi
deli strukture iz proteinov donorjev na osnovi ProBiS algoritma, ki omogoča odkrivanje
lokalno podobnih strukturnih vzorcev fizikalno-kemijskih lastnosti na površinah proteinov.
Donorje bomo izbirali med človeškimi proteini iz Protein Data Bank. Naš cilj je terapevtski
protein spremeniti tako, da bo pridobil želene lastnosti donorja, to je, da se na primer ne bo
vezal na Fcγ receptorje, hkrati pa bo stabilen in ne bo imunogen. Računalniški pristop bomo
razvili in umerili ter prvikrat uporabili na terapevtskem proteinu – ipilimumab – monoklonskem
protitelesu na osnovi človeškega IgG1, ki se uporablja za zdravljenje melanoma, pa tudi
drugih rakov. Orodja bodo prosto dostopna širši raziskovalni skupnosti in bodo omogočala
načrtovanje proteinov z novimi želenimi lastnostmi.
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 Tlymphocyte-
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.