Natural products are an important resource for cancer therapy, with highly potent and diverse anticancer activities. Natural product biosynthesis is well comprehended, however the evolutionary principles governing the alteration of enzymatic assembly lines to yield molecules with activity toward distinct various cellular targets are not understood. This gap in knowledge hinders efforts to synthetically combinatorialize assembly lines to yield “unnatural” natural products with important or hybrid activity toward up-regulated targets in cancer. Furthermore, natural products did not evolve in the context of mammalian systems and would benefit from a delivery mechanism to cancerous cells to improve their ability to generate successful clinical outcomes. Consequently, natural products were linked to antibodies targeted to cell surface proteins up-regulated on cancer cells, generating antibody-drug conjugates (ADC). The conjugation methodology is problematic by yielding ADCs with varying numbers of drugs loaded per antibody. This lack of batch-to-batch standardization limits our ability to completely evaluate the safety profiles and efficacy of ADCs and determine proper dosages for patients. In this research, light was shed on biosynthetic evolutionary changes through the study of the antimycin-type family of depsipeptides, specifically demonstrating that modular insertions or deletions lead to natural product structural diversification. Additionally, a novel biosynthetic enzymatic method was established to site-selectively conjugate natural products to antibodies in order to facilitate the development of more sophisticated cancer therapies. / Thesis / Master of Science (MSc)

This diploma thesis deals with the synthesis of conjugates of cyclodextrin with the anticancer drug 5-fluorouracil and folic acid, which works as a targeting group. 5-Fluorouracil is connected to cyclodextrin via an acid-labile linker, which is expected to be cleaved in decreased pH in the proximity of malignant tissue or in the endosome. Malignant tissue also overexpresses receptor for folic acid, and this phenomenon is used for targeted delivery of therapeutic agents. Cyclodextrins are cyclic oligosaccharides, which are known for their ability to complex various compounds into their hydrophobic cavity and increase solubility, stability and bioavailability of these compounds. A synthetic approach for the preparation of conjugate of cyclodextrin with 5-fluorouracil and folic acid was designed and the conjugate was subsequently synthesized. Key words: cyclodextrin, fluorouracil, targeting group, folic acid, drug delivery

Les Thrombopénies Induites par l’Héparine (TIH) sont une complication sévère des traitements par l’héparine dues à des IgG qui ciblent le facteur plaquettaire 4 modifié par l’héparine (FP4/H) et induisent une activation cellulaire via FcγRIIA, conduisant à des complications thrombotiques. Nous avons caractérisé 5B9, IgG1 monoclonale chimérique anti-FP4/H mimant parfaitement les anticorps de TIH et qui est donc un excellent outil pour étudier la physiopathologie des TIH. La pathogénicité des anticorps (Ac) de TIH implique leur fixation aux FcγR. Nous avons montré que le clivage de la région charnière des IgG de TIH par IdeS inhibe ces interactions IgG-FcγR et supprime la pathogénicité des Ac. Nous avons aussi construit un Antibody-Drug Conjugate (ADC) antithrombotique, en bioconjuguant le tirofiban (inhibiteur de l’agrégation plaquettaire) et 5B9 déglycosylé grâce à un linker clivable par la thrombine, protéase générée en excès lors d’une TIH. / Heparin Induced Thrombocytopenia (HIT) is a rare but severe complication of heparin treatments. HIT is due to IgG antibodies specific to platelet factor 4 modified by heparin (PF4/H), which activate blood cells, (especially platelets) after binding to FcγRIIA, this process explaining frequent thrombotic complications. We characterized 5B9, a chimeric IgG1 targeting PF4/H and which fully mimics human HIT antibodies. Therefore, 5B9 is a perfect tool for studying the physiopathology of HIT. IgG antibodies to PF4/H are pathogenic by interacting with FcγR. In this regard, we showed that cleavage by IdeS, a bacterial protease, of the hinge of anti-PF4/H IgG, fully suppressed their pathogenicity. Furthermore, we designed an antithrombotic Antibody-Drug Conjugate that combined tirofiban, a GPIIbIIIa inhibitor with deglycosylated 5B9 using a thrombin cleavable linker.

La plupart des médicaments développés pour les maladies du SNC n’atteignent pas leur cible en raison des propriétés uniques de la BHE, nécessitant la mise en place de stratégies de délivrance comme l'utilisation d'un processus physiologique, le RMT. Des peptides ciblant le LDLR (exprimé à la BHE et impliqué dans ces processus) ont été développés. Les objectifs de cette thèse ont été de caractériser le trafic intracellulaire et la capacité de transport de différentes formes de ces peptides dans différents modèles in vitro y compris dans un modèle de BHE.Les résultats obtenus dans une lignée cellulaire surexprimant le LDLR tagué GFP par imagerie en fluorescence montrent que les différentes formes de ces peptides lient le LDLR à la membrane plasmique d’où ils sont internalisés et adressés aux lysosomes sans interférer avec l’endocytose des LDL. Ils permettent l’adressage aux lysosomes de petites molécules (fluorochrome) et de protéines qui leur sont fusionnées, ces résultats indiquent qu’ils pourraient être utilisés pour cibler des molécules thérapeutiques aux lysosomes de cellules exprimant les LDLR. Dans le modèle in vitro de BHE, les peptides sont internalisés via le LDLR à partir du pôle apical et suivent un transport intracellulaire similaire aux LDL, étant déroutés de la voie de dégradation vers les lysosomes pour être transportés jusqu’au compartiment abluminal comme précédemment décrit pour le LDL et la transferrine. Ces données indiquent donc que les peptides ciblant le LDLR sont des candidats vecteurs intéressants pour compléter/améliorer le panel de peptide/anticorps existant et permettre le ciblage et le transport de molécules thérapeutiques à travers la BHE. / Many drugs are ineffective in treating CNS diseases due in part to unique properties of the BBB, requiring the establishment of delivery strategies such as the use of a physiological process, as the RMT. Peptides targeting the LDLR (expressed in the BBB and involved in these processes) have been developed. The objectives of this thesis were to characterize the intracellular traffic and transport capacity of different shapes of these peptides in various in vitro models including a model of BBB.The results obtained in a cell line overexpressing the LDLR tagged GFP by fluorescence imaging shows that the various forms of these peptides bind plasma membrane LDLR, where they are internalized and sent to lysosomes without interfering with LDL endocytosis. They allow lysosomal targeting of small molecules (fluorochrome) and proteins that are fused to them. These results indicate that it might be used to target therapeutic compounds to cells expressing LDLR lysosomes. In the in vitro BBB model, the peptides are internalized via the LDLR from the apical pole and follow a similar intracellular transport than LDL, being diverted from the lysosomal degradation pathway to be transported to the abluminal compartment as previously described for LDL and transferrin. These data indicate that the LDLR-targeting peptides seems useful vectors candidates to complete/improve the existing peptide/antibodies panel and allow the targeting and the transport of therapeutic molecules through the BBB.

No description available.

Affibodymolekyler är små affinitetsproteiner (6.5 kDa) som föreslås kunna ersätta monoklonala antikroppar i terapeutiska tillämpningsområden, exempelvis i antikropp-läkemedelskonjugat (ADCs) som kan navigera sig fram till biomarkörer som är uttryckta på cancerceller. Affibody-läkemedelskonjugat (AffiDC) kan användas för att målsöka just sådana överuttryckta proteiner, samtidigt som de erbjuder goda egenskaper, såsom snabb transportering och spridning i kroppen, och effektiv penetrering genom tumörer. Dessa AffiDC:er skulle kunna användas inom riktad cancerterapi for de cancersjukdomar som fortfarande är i behov av cancerhämmande behandlingar, såsom urotelial cancer.  Den här studien föreslog tillämpning av ABD-kopplade affibodymolekyler för att målsöka ett nytt målprotein som har visats vara överuttryckt i flera olika cancersjukdomar, exempelvis bröst-, pankreas- och urotelial cancer. Affibodykandidater mot målproteinet har valts ur ett rekombinant bibliotek med 1×1011 transformanter som uttrycks med hjälp av en så kallad metod med E. coli celldisplay där affibodymolekylen visas på cellens ytmembran. De slutliga kandidaterna var sedan identifierade och biokemiskt karaktäriserade i in vitro-studie på människoceller, som visade att två av kandidaterna verkade binda till cancercellinjerna BT-474 och MCF-7 med KD omkring 10 till 100 nM. / Affibody molecules are small affinity proteins (6.5 kDa) suggested to substitute monoclonal antibodies in therapeutic applications, e.g., antibody-drug conjugates (ADCs) targeting biomarker proteins expressed on cancer cells. An affibody-drug conjugate (AffiDC) could be used to target these types of overexpressed proteins on cancer cells while offering attractive properties, such as rapid transportation and distribution in the body, as well as efficient tumour penetration. These AffiDCs could be used as a targeted cancer therapy for cancers that are yet to be treatable and curable, like urothelial cancers.  This study suggested the use of ABD-fused affibodies to target a novel cancer protein that has been shown to be overexpressed on cancer cells, including breast, pancreatic and urothelial cancer. Affibody candidates toward this novel target were selected from a recombinant library, of 1×1011 transformants, that is expressed using E. coli cell display system. The final candidates were subsequently biochemically characterized and assessed for affinity for the target. Three affibodies were finally identified and assessed in in vitro studies on mammalian cells, revealing two affibodies that appear to bind to the cell lines BT-474 and MCF-7 with KD ranging 10 to 100 nM.

No description available.

Bone Cancer Research Trust / The full text will be available at the end of the embargo: 6th October 2027

京都大学 / 0048 / 新制・論文博士 / 博士(工学) / 乙第13145号 / 論工博第4163号 / 新制||工||1687(附属図書館) / (主査)教授 森 泰生, 教授 浜地 格, 教授 梅田 眞郷 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Surgery-induced myeloid derived suppressor cells (sxMDSC)s mediate postoperative suppression of Natural Killer (NK) cells, which enables postoperative cancer recurrence and metastases. Currently, no therapeutics against sxMDSCs have been developed. Recent research has identified that the myeloid-restricted PI3K isoform (PI3Kγ) mediates MDSC activity. I targeted PI3Kγ in sxMDSCs as a therapeutic to reduce postoperative NK cell suppression and metastatic burden. Additionally, I investigated the efficacy of a sxMDSC-specific antibody-drug conjugate (ADC) with a PI3Kγ inhibitor payload. Pharmacological inhibition of PI3Kγ in sxMDSCs led to reduced AKT phosphorylation and reduced suppression of NK cytotoxicity in human and murine models. PI3Kγ inhibition also reduced postoperative metastatic burden. Despite the novelty of the sxMDSC-specific ADC, it didn’t provide considerable benefits in reducing NK cell suppression compared to the unconjugated PI3Kγ inhibitor. However, this is a “first iteration” in what could be a powerful approach to targeting sxMDSCs, thereby preventing postoperative metastatic burden.