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Imunoterapija raka s celicami CAR-T predstavlja uspešno alternativo klasičnemu zdravljenju raka. Uspehe dosega predvsem pri hematoloških oblikah raka, kot so različni limfomi in akutna limfoblastna levkemija, medtem ko so solidni tumorji še vedno izziv. K slabšemu učinku terapije doprinesejo predvsem T-celična izčrpanost in senescenca ter inhibitorno tumorsko mikrookolje solidnih tumorjev, zato se išče rešitve v smeri dodajanja domen himernemu antigenskemu receptorju, ki bi vplivale na učinkovitost terapije- tako na večjo aktivnost celic kot tudi na njihovo daljšo obstojnost v telesu, pri čemer ne bi poslabšali lastnosti imunoterapije CAR-T. Pri tem si pomagamo z orodji sintezne biologije. Naš cilj je bil ustvariti receptor CD19 CAR, ki bi poleg klasičnih elementov vseboval še domene izhajajoče iz adapterskega proteina MyD88, ki igra pomembno vlogo v naravni imunosti. Z različnimi metodami smo dobili vpogled v vpliv tako spremenjenega receptorja na aktivnost modificiranih celic ob ustrezni stimulaciji, na izražanje receptorja in na izražanje inhibitorja ključnega transkripcijskega faktorja NF-κB, IκBα. Opazili smo večjo aktivnost, izražanje receptorja na membrani in različno izražanje IκBα glede na čas stimulacije. CAR-T immunotherapy for cancer represents a successful alternative to classic cancer treatment. It is very successful with the haematological malignancies such as different lymphomas and acute lymphoblastic leukemia, however not so much with solid tumors, which still represent a challenge. Contributors that lower response rates are mainly T-cell exhaustion, senescence and inhibitory tumor microenvironment. That is why researchers are looking for a solution by adding domains to chimeric antigen receptor which would affect efficiency of the therapy- meaning higher activity and longer persistence, where negative aspects of the CAR-T immunotherapy would not be increased. To modulate CAR-T immunotherapy synthetic biology tools are used. Our aim was to create CD19 CAR receptor which would express domains from MyD88 adapter protein, a key mediator of innate immunity, in addition to traditional receptor elements. By using different methods we got an insight into the effect of such a receptor on activity of modified cells when stimulated correctly. We also got an insight into expression of receptor and expression of inhibitor of the key transcription factors NF-κB, IκBα. We observed higher activity, receptor expression on the cell membrane and different IκBα expression at different stimulation time.

Imunoterapija raka je skupina sodobnih terapevtskih pristopov za tarčno zdravljenje malignih obolenj. Zelo uspešna imunoterapevtska strategija je zdravljenje s celicami CAR-T. Zdravljenje poteka z avtolognimi limfociti T, v katere se vstavi genski zapis za receptor CAR, celice pa se nato znova injicira v pacienta. Celice CAR-T prepoznajo tumorski antigen, kar povzroči T-celično aktivacijo in uničenje rakavih celic. CAR-T celične terapije so odobrene za klinično rabo, vendar so uspehi zaenkrat omejeni le na nekatere vrste krvnih rakov. V okviru magistrskega dela smo razvili nov dizajn receptorjev CAR, ki temeljijo na uporabi verige CD3ε, s čimer smo želeli doseči kooperativnost med CAR in nativnim TCR. Pripravili smo pet različnih genskih konstruktov CAR in potrdili njihovo izražanje na površini limfocitov T. Novi receptorji CAR so ob stimulaciji s tarčnim antigenom sprožili T-celično aktivacijo. Interakcija med receptorjem CAR in nativnim TCR je bila bolj izrazita pri inovativnih receptorjih CAR kot pri konvencionalnih CAR. Za preučevanje mehanizma interakcije med CAR in TCR smo z uporabo metode CRISPR-Cas9 začeli razvijati celično linijo z izbitim genom za CD3ε. Dizajn receptorjev CAR na osnovi verige CD3ε bo služil za nadaljnji razvoj in optimizacijo CAR-T celične terapije. Cancer immunotherapy is a group of modern therapeutic approaches for targeted treatment of malignancies. CAR-T cell therapy is a very successful immunotherapeutic strategy where a genetic material for the CAR receptor is inserted into the patient's lymphocytes and then reinfused back into the body. CAR-T cells are able to recognize tumour antigen, trigger T-cell activation, and subsequently kill cancer cells. While CAR-T cells are already approved for clinical use, their success is limited to certain blood cancers. We developed a new design of CAR receptor that is based on the CD3ε chain, through which we tried to achieve cooperation between CAR and TCR. We designed and prepared five CAR constructs and showed their surface expression. We confirmed that the receptors were able to trigger T-cell activation upon stimulation with the target antigen. Interaction between CAR and TCR was more pronounced in new designs of CARs than in conventional CARs. We have also started developing a cell line with knocked-out CD3ε gene for studying the mechanism of TCR-CAR interaction. CARs based on the CD3ε chain will serve as a platform for further development and optimisation of CAR-T cell therapies.

Indolamin 2,3-dioksigenaza 1 (IDO1) je poglavitni encim kinureninske metabolne poti, kjer katalizira pretvorbo L-triptofana v N-formilkinurenin. Za večino vrst rakavih obolenj je značilno povišano izražanje encima, kar je v tesni povezavi s slabo prognozo in nižjo možnostjo preživetja. IDO1 je vpleten v zaviranje efektorske funkcije celic T in ubijalk, diferenciacijo in aktivacijo imunosupresivnih regulatornih limfocitov T ter celic mieloidne supresorske vrste, kar promovira razrast tumorskih celic, omogoča neovaskularizacijo in zmanjšuje učinkovitost imunoterapij. Rezultati prvih in drugih faz kliničnih vrednotenj zaviralcev IDO1 so obetali protitumorno delovanje, a zaenkrat še nimamo potencialne učinkovine, ki bi prestala vse klinične faze. V sklopu magistrske naloge smo želeli z uporabo ekspresijskega sistema Escherichia coli pridobiti IDO1, ki bi izkazoval ekvivalentno aktivnost v primerjavi s komercialno dostopnim encimom in sintetizirati nove zaviralce IDO1 ter ovrednotiti njihovo zaviralno aktivnost. Z optimizacijo sestave gojišča in pogojev gojenja smo skušali zagotoviti čim večje izražanje. Celicam smo dodali različne prekurzorje za sintezo hema in primerjali izkoristek gojenja po 24 h in 48 h v prisotnosti oz. odsotnosti induktorja. S poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata smo potrdili istovetnost produkta in pretežno zastopanost v topni obliki, nakar smo protein izolirali in očistili s kovinsko-kelatno afinitetno kromatografijo in gelsko filtracijo. Koncentracijo očiščenega encima smo določili spektrofotometrično pri 280 nm, še bolj pa nas je zanimala vrednost razmerja absorbanc A404/A280, ki korelira z deležem proteina, na katerega je vezan hem, kar vpliva na encimsko aktivnost. Največ rekombinantnega encima smo uspeli pridobiti, če smo bakterijam v gojišče dodali 5-aminolevulinsko kislino in z gojenjem nadaljevali še 24 h po indukciji izražanja. Aktivnost rekombinantnega encima IDO1 je bila kar petkrat nižja od komercialnega, verjetno na račun odsotnosti reducenta po zadnjem kromatografskem čiščenju. Sintetizirali smo tri končne spojine na osnovi 2-(3-(4-fluorofenil)-4-oksoizoksazolo[5,4-d]pirimidin-7(4H)-il)-N-fenilacetamidnega skeleta, ki so se razlikovale v substituentu na položaju meta fenilnega obroča, vezanega na N-atomu acetamida. Zaviralno delovanje končnih spojin smo vrednotili s fluorescenčnim testom, pri katerem se je kot najmočnejši zaviralec z IC50 vrednostjo 55.1 µM izkazala spojina 10 s trifluorometilno skupino. Čeprav rekombinantni encim ni dosegel želene aktivnosti, pa smo z našim raziskovalnim delom pomembno prispevali k temu, da bo v prihodnje možno pridobiti kristalno strukturo in na osnovi lastnosti vezavnega mesta načrtovati še močnejše zaviralce. Indoleamine 2,3-dioxygenase 1 (IDO1) is a major enzyme of the kynurenine pathway, where it catalyzes the conversion of L-tryptophan to N-formylkynurenine. Higher expression of the enzyme is observed in the majority of cancer related diseases, which is closely associated with bad prognosis and lower survival rates. IDO1 suppresses effector function of T and natural killer cells, differentiation and activation of immunosuppressive regulatory T lymphocytes and myeloid derived suppressor cells, which promotes spread of tumour cells, neovascularization and diminishes effectiveness of immunotherapies. Results of the first and the second phases of clinical trials showed promising antitumour activity however, up to date no IDO1 inhibitors have passed all clinical phases. As a part of master's thesis we wished to express in Escherichia coli, isolate and purify IDO1 that would show equivalent activity to the commercially available enzyme synthesize novel IDO1 inhibitors and evaluate their inhibitory activity. With optimization of growth medium composition and culture condition we tried to secure higher expression. Cells were provided with heme precursors for its synthesis and we compared expression yields after 24 and 48 hours of cultivation in the presence/omittance of an inductor. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed to confirm identity of the product and its presence in soluble form. In addition, the protein was isolated and purified with immobilized metal affinity chromatography and gel filtration chromatography. Concentration of a purified enzyme was determined spectrophotometrically at 280 nm, but of greater importance was the absorbance ratio A404/A280, which correlates with proportion of cofactorial heme occupation, which influences enzyme activity. Highest IDO1 expression was achieved when the growth medium was supplemented with 5-aminolevulinic acid and bacteria were grown for 24 hours upon recombinant protein induction. The activity of produced enzyme was 5-fold lower compared to the commercial one, most likely due to the fact that the reducent was not added after the last purification step. We synthesized three final compounds based on 2-(3-(4-fluorophenil)-4-oxoisoxasolo[5,4-d]pyrimidine-7(4H)-yl)-N-phenylacetamide scaffold, where we added different substituents on the meta position of the phenyl ring, attached to the N-atom of acetamide group. Inhibitory activity of final compounds was determined with a fluorescence-based assay, where the compound 10 with trifluoromethyl substituent exhibited the most potent inhibitory activity, with the IC50 value of 55.1 µM. Although we failed to produce the enzyme with equivalent activity to the commercial counterpart, our contribution for future research cannot be neglected as it might soon be possible to obtain new crystal structures and design novel more potent inhibitors on the basis of binding site characteristics.

Indolamin 2,3-dioksigenaza 1 (IDO1) je hem vsebujoči citosolni encim, ki katalizira prvo stopnjo pretvorbe triptofana, pri tem kot glavni toksični metabolit nastaja kinurenin. Pomanjkanje triptofana in povečana koncentracija kinurenina v tumorskem mikrookolju vpliva na tri signalne poti v celici, kar vodi do prekritja tumorskih celic imunskemu sistemu in s tem do napredovanja bolezni. K napredovanju bolezni prav tako prispeva z IDO1 povzročena neovaskularizacija. Iz teh razlogov predstavlja IDO1 zanimivo potencialno tarčo v imunoterapiji raka. Trenutno je v kliničnih študijah kar nekaj zaviralcev IDO1. Študije pa kažejo predvsem na sinergistično delovanje v kombinaciji z uveljavljenimi načini zdravljenja raka. Namen naše naloge je bil na osnovi 3-fenilizoksazolo[5,4-d]pirimidin-4(5H)-ona sintetizirati in biokemijsko ovrednotiti nove zaviralce IDO1, od katerih bi vsaj eden zaviral delovanje encima v nizkem mikromolarnem območju. Prav tako pa smo želeli v sklopu magistrske naloge optimizirati s stališča izkoristkov in čistosti intermediatov najbolj problematičen korak splošne sintezne poti, ki ga predstavlja sinteza izoksazol-4-karboksamida. Sinteza novih zaviralcev IDO1 je temeljila na večstopenjski sintezni poti, s katero smo pripravili devet končnih spojin, ki smo jim z različnimi analitskimi tehnikami potrdili istovetnost in čistost. Z biokemijskim testiranjem na osnovi merjenja fluorescence pa smo ovrednotili njihovo zaviralno delovanje na IDO1. V nizkem mikromolarnem območju so svoje vrednosti IC50 imele tri spojine, in sicer 31, 32 in 33. Kot najmočnejši zaviralec IDO1 pa se je izkazal 2-(3-(3,4-difluorofenil)-4-oksoizoksazolo[5,4-d]pirimidin-5(4H)-il)-N-(4-nitrofenil)acetamid (31) z vrednostjo IC50 68,48 µM. Na osnovi analize molekulskega sidranja spojine 31 v aktivno mesto encima smo poizkušali napovedati potencialne interakcije, ki jih spojina 31 tvori z aminokislinskimi ostanki v aktivnem mestu. Na osnovi naših rezultatov lahko zaključimo, da spojina 31 predstavlja pomembno izhodišče za nadaljnje raziskave in razvoj močnejših novih zaviralcev IDO1 kot potencialnih protitumornih učinkovin. Indolamine 2,3-dioxygenase 1 (IDO1) is a heme-containing cytosolic enzyme that catalyzes the first step of tryptophan metabolism, which leads to the formation of kynurenine as the major toxic metabolite. Lack of tryptophan and increased concentration of kynurenine in the tumor microenvironment affects three signaling pathways in the cell, leading to the immune escape of tumor cells and disease progression. IDO1-induced neovascularization also contributes to disease progression. For these reasons, IDO1 represents an interesting potential target in cancer immunotherapy. There are currently quite a few IDO1 inhibitors in clinical trials. So far the findings show a synergistic effect in combination with established treatments for cancer. The aim of our thesis was to synthesize and biochemically evaluate novel IDO1 inhibitors on the basis of 3-phenylisoxazolo[5,4-d]pyrimidin-4(5H)-one in order to obtain at least one inhibitor in the micromolar range. Furthermore, we also wanted to optimize the yields and purity of intermediates of the most problematic step of the general synthetic pathway, which is the synthesis of isoxazole-4-carboxamide. The synthesis of new IDO1 inhibitors was based on the multi-step synthetic route, which was used to prepare nine final compounds. Their identity and purity was confirmed by various analytical techniques. Biochemical evaluation based on fluorescence measurement was used to determine their inhibitory activity on IDO1. Three compounds (31, 32 and 33) had their IC50 values in low micromolar range. The most potent inhibitor of IDO1 was 2-(3-(3,4-difluorophenyl)-4-oxoisoxazolo[5,4-d]pyrimidin-5(4H)-yl)-N-(4-nitrophenyl)acetamide (31) with an IC50 value of 68,48 μM. Based on the molecular docking of compound 31 into the enzyme's active site we predicted potential interactions of 31 with amino acid residues. According to results obtained for 31 it represents an important starting point for further research and development of more potent new IDO1 inhibitors as potential anticancer agents.

Razvoj terapevtskih postopkov rakavih obolenj se zaradi kompleksnosti slednjih nagiba k usmerjenim in personaliziranim adaptivnim celičnim terapijam. Mednje sodijo celice CD19 CAR-T, pri čemer pacientovim limfocitom T vstavimo receptor CAR, specifičen za B-limfocitni antigen CD19. Terapija je uspešna pri B-celičnih malignih obolenjih, vendar so prisotni stranski učinki (SIRS, CRS) zaradi katerih je potrebno uravnavanje aktivnosti celic CAR-T. Z namenom regulacije celic CAR-T smo na osnovi transkripcijskega faktorja NFAT in heterodimerizacijskih domen ABI-PYL1, DmrA-DmrC in GAI-GID1 pripravili regulatorni sistem, ki celice aktivira ali represira. V celicah HEK293T in Jurkat smo potrdili, da ob dodatku ustreznih induktorjev (abscizinska kislina, rapamicin in giberelin) pride do heterodimerizacije konstruktov in da se NFAT učinkovito veže na vezavna mesta za DNA, pri čemer zanje tekmuje z endogenim NFAT. Hkrati smo pokazali, da heterodimerizacijski sistemi robustno aktivirajo celice Jurkat z receptorjem CD19-CAR ob prisotnosti celic, ki izražajo tarčni antigen (celice Raji). In recent years, cancer immunotherapy has been leaning towards personalized, T-cell mediated therapy, due to the complex background of the disease. CD19 CAR-T cells represent a cure for hematologic malignancies. The therapy, albeit successful, does not avoid side effects such as SIRS and CRS and thus requires a development of a robust and reliable regulation of CAR-T cells. With CAR-T regulation in mind, we developed a regulatory system, based on transcription factor NFAT and heterodimerization domains ABI-PYL1, DmrA-DmrC, GAI-GID1, which either activates or represses the cells in question. We show that in the presence of chemical inductors (abscisic acid, rapamycin and gibberellin) the constructs undergo heterodimerization and that engineered NFAT, competing with endogenic NFAT, binds to its regulatory site of both reporter gene (shown in HEK293-T cells) or IL-2 (shown in Jurkat cells). We also confirm, that heterodimerization systems work in cells with expressed CD19-CAR and in presence of target antigen on the surface of Raji cells.

Terapevtska monoklonska protitelesa se uporabljajo za zdravljenje različnih rakavih obolenj, hkrati pa se njihov proti tumorski učinek lahko še izboljša z dodatkom efektorskih molekul, kot so na primer superantigeni (SAg). Superantigeni so bakterijski ali virusni toksini s sposobnostjo aktivacije velikega števila celic T. Njihova bistvena lastnost je, da se vežejo neposredno na poglavitni kompleks tkivne skladnosti razreda II (MHCII), izražen na antigen predstavitvenih celicah, kar vodi v aktivacijo celic T preko celičnega receptorja T (TCR). Ena izmed pomanjkljivosti terapevtske uporabe superantigenov, spojenih s protitelesi, specifičnimi za tumorske antigene, je sistemska aktivacija T-celičnega odziva, ki je posledica visoke afinitete superantigena do kompleksa MHCII. Naša ideja je bila razdeliti superantigen stafilokokni enterotoksin A (SEA) iz Staphylococcus aureus na dva neaktivna fragmenta, ki se sestavita v biološko aktivno obliko le, ko prideta v neposredno bližino. V študiji smo vzpostavili hitro in enostavno metodo za odkrivanje učinkovitih cepljenih različic superantigena, ki temelji na spojitvi fragmentov SEA s peptidi, ki tvorijo obvite vijačnice. Potrdili smo, da je superantigen SEA mogoče razdeliti na dva neaktivna fragmenta, ki se spontano ne sestavita v biološko aktivno obliko, ampak ju morata peptida, ki tvorita heterodimer, pripeljati v neposredno bližino. Kot dokaz principa uporabe za namene imunoterapije raka smo učinkovito različico cepljenega SEA spojili z enoverižnim variabilnim fragmentom monoklonskega protitelesa proti antigenu CD20 (scFv-CD20). Potrdili smo, da sta fragmenta SEA le ob vezavi na tarčni antigen CD20 preko scFv-CD20, dovolj blizu skupaj, da se sestavita v biološko aktivno obliko, ki aktivira limfocite T. Fuzijski proteini, pripravljeni v doktorskem delu, imajo potencialno terapevtsko vrednost za zdravljenje rakavih obolenj, saj smo potrdili, da je njihovo delovanje usmerjeno le proti tumorskim celicam, brez vpliva na netarčne celice. Antibody-based therapeutics that target surface antigens expressed on tumor cells are successfully used for treatment of different types of cancer. Although unconjugated monoclonal antibodies are efficient, conjugating different effector molecules, such as superantigens (SAg) to monoclonal antibodies can enhance their clinical utility. SAg are bacterial or viral toxins and one of the most potent activators of T cells, which directly bind to major histocompatibility complex class II (MHC class II) on antigen presenting cells and activate T cells through T cell receptor (TCR). Strong T cell activation is also one of the main weaknesses of this strategy as it may lead to systemic T cell activation, due to high affinity of superantigens to MHC class II. To overcome the limitation of conventional antibody-superantigen fusion proteins, we have split a staphylococcal enterotoxin A (SEA) from Staphylococcus aureus into two fragments, individually inactive, until both fragments came into close proximity and reassemble into a biologically active form capable of activating T cell response. We developed a fast and simple screening method based on a fusion between SEA and coiled-coil heterodimers that enabled a detection of functional split SEA designs. In this study we showed that it is possible to split SEA into two inactive fragments that do not reassemble spontaneously, but when fused with heterodimerizing domains, which bring fragments into proximity, split SEA can reassemble into a biologically active form. As proof-of-principle of cancer immunotherapeutic the split SEA design that demonstrated efficacy in fusion with coiled-coil dimer forming polypeptides, was fused to a single chain antibody specific for tumor antigen CD20 (scFv-CD20). This design selectively activated T cells by split SEA-scFv fusion binding to target antigen CD20. Prepared fusion proteins have a potential therapeutic value for cancer treatment, as they specifically bind to tumor cells and have no effect on non-target cells.