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Student Number : 9903022H MSc Dissertation School of Chemistry Faculty of Science / Cancer is characterised by the unconstrained growth of cancerous cells, which damages the healthy cells and ultimately the tissue of the host. Chemotherapy forms an essential component in the treatment of this disease, however most anti-tumour drugs suffer from various deficiencies, e.g. increased toxicity, reduced serum half life and poor water solubility. The focus of this project was to address some of these deficiencies by conjugating selected drugs to a water-soluble polymeric carrier. Selected water-soluble biodegradable carriers were synthesized. Copolyaspartamides, polyamidoamines and polyamides were obtained by condensation polymerisation, Michéal–type addition polymerisation and ester amine base-catalysed polymerisation. The nascent water soluble polymers were used to conjugate platinum, ferrocene and tetramethylmelamine derivative, respectively. The percentage drug in each polymer drug conjugate was determined by considering the mass of the drug in the conjugate as a percentage of the total mass of the drug-polymer conjugate. Platinum was linked to the carrier via polymer attached amine, carboxyl and hydroxyl ligands. Platinum content of the conjugates ranged from 7 to 11 % by mass. The ferrocenylation agent, 4-ferrocenylbutanoic acid, and the tetramethylmelamine derivative, 3-(4,6-bis(N,N-dimethylamino)-1,3,5-triazacyclohexatrien-2-yl) propanoic acid was polymer-bound by amidation reactions. Iron content of the ferrocence conjugated ranged from 2 to 12 % by mass. While the drug content based on tetramethylmelamine in the 3-(4,6-bis[N,N-dimethylamino]-1,3,5-triazacyclohexatrien-2-yl) propanoic acid polymer conjugate ranged from 8.4 to 8.6 % by mass. There was a preliminary attempt to coconjugate both, 4-ferrocenylbutanoic acid and 3-(4,6-bis[N,N-dimethylamino]-1,3,5- triazacyclohexatrien-2-yl) propanoic acid to the same polymer. This co-conjugate contained 2.9 % iron and 3.4 % tetramethylmelamine by mass.

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Pancreatic ductal adenocarcinoma (PDAC) is the most lethal common cancer in the United States: in 2017 there will be around 54,000 new cases and 43,000 patient deaths. (SEER, 2017) The high mortality of PDAC is related to late disease presentation and aggressiveness; nearly 52% of patients present with metastatic disease at the time of diagnosis. (SEER, 2017) Current treatments have marginal improvements on survival, with the most efficacious treatment, gemcitabine and nab-paclitaxel, having a median survival of 12.2 months. (Wu 2018) Failure of current PDAC treatments is attributed to the inefficacy of systemic chemotherapeutics and the development of resistance. (Rahib, 2014) The Dual Endothelin1/Signal PeptideVEGF receptor (DEspR), represents a promising therapeutic target for the treatment of PDAC: it is a highly expressed, specific tumor antigen, which is involved in tumor vasculogenesis and cancer stem cell (CSC) survival. DEspR is a developmentally crucial receptor, responsible for early angiogenesis and neural crest migration, with minimal expression in normal adult tissue. In vitro and in vivo studies of anti-DEspR therapy in PDAC have shown efficacy in decreasing CSC survival, tumor angiogenesis, and improving overall survival in xenograft models of PDAC, with anti-DEspR therapy being a promising candidate for clinical use. Furthermore, anti-DEspR therapy seems to augment chemotherapeutic therapy in vitro and in vivo, suggesting that a DEspR-targeted antibody drug conjugate (ADC) would be highly effective. ADCs are a re-emerging drug class with significant promise. Initial failures of ADCs in clinic were related to poor antigen specificity and failures in drug conjugation chemistry to minimally impact the antibody. To develop our ADC, I have developed a novel method of site-specific conjugation that relies on a novel method of supramolecular assembly. My system employs two specific protein sequences that do not self-interact, and tightly assemble through coulombic and hydrophobic interactions, allowing site-specific, stoichiometric self-assembly. To facilitate stable drug delivery, I have synthesized a novel enzymatically cleavable tyrosine-clickable linker, which prevents drug release prior to tumor delivery. Both further investigation into the efficacy of anti-DEspR therapy, and the development of a stoichiometric, site-specific, stable method for drug loading will provide an advancement in anti-cancer therapy.

Es ADCs (Antibody-Drug Conjugates) correspondent à une nouvelle stratégie thérapeutique anti-tumorale particulièrement prometteuse. Néanmoins, les ADCs actuellement utilisés en clinique sont obtenus par conjugaisons chimiques, resultant en des mixtures hétérogènes impactant négativement leurs pharmacocinétiques et leurs performances in vivo.Récemment, différentes strategies de couplage site-spécifique ont été développées afin de réduire cette hétérogénéité. Dans cette thèse, nous rapportons le développement d’une nouvelle technologie CovIsoLink™ (Covalently Isopeptide Crosslinking) permettant la génération d’ADCs par utilisation de nouveaux peptides glutamine Q-Tag présentant des affinités optimisées par rapport à des peptides disponibles (ZQG, LLQG) pour une enzyme bactérienne la transglutaminase (mTG).La preuve de concept de cette technologie a été réalisée par insertion de ces peptides Q-Tag en C-ter de la région codant pour la chaine lourde des anticorps anti-HER2 (Trastuzumab). Nous avons ainsi pu démontrer la conjugaison homogène et reproductible de différentes drogues sans contamination par des chaines d’anticorps non conjuguées. Nous avons pu montrer que l’immunoréactivité et la capacité d’internalisation de ces ADCs n’étaient pas altérées par la conjugaison et qu’ils présentaient in vitro et in vivo, des propriétés de lyse de cellules tumorales similaires au Trastuzumab emtansine (Kadcyla®), actuellement en clinique. Par ailleurs, afin de généraliser notre technologie à différents formats d’anticorps nous avons générés des fragments Fab et scFv et évalué leur fonctionnalité. Ainsi, nous avons pu prouver que l’utilisation de nouveaux peptides optimisés Q-Tag substrat de la transglutaminase permettait une stratégie de couplage alternative plus homogène par couplage de différentes molécules sans espèce contaminante non couplée / Antibody-drug conjugates (ADCs) are a powerful class of therapeutic agents, demonstrating success in the treatment of several malignancies. The currently approved ADCs are produced by chemical conjugations and exist as heterogeneous mixtures that negatively influence the pharmacokinetics and in vivo performance. Recently many of site-specific conjugation technologies have been developed to reduce heterogeneity and batch-to batch variability. Microbial transglutaminase (mTG) has been demonstrated as efficient tool for site-specific conjugation. In this thesis we report the development CovIsoLink™ (Covalently Isopeptide Crosslinking) technology for the generation of homogenous immunoconjugates using a novel glutamine donor peptides (Q-tag) with improved affinity compared to the known peptides (ZQG, LLQG). As a proof of concept, the peptides sequences were engineered into the heavy chain C-terminal of Trastuzumab antibody. We demonstrated the reproducible and homogeneous conjugation of Q-tagged Trastuzumab with different payloads, without any unconjugated species. The ADCs were evaluated in series of in vitro and in vivo assays. We confirmed that the immunoreactivity and internalisation are not altered by the conjugation. Furthermore similar in vitro and in vivo tumor cell killing potency was demonstrated than Trastuzumab emtansine (Kadcyla®), which is already used in the clinic. Morover we extend our site-specific conjugation technology to antibody fragments (Fab and scFv), evaluating their functionality by conjugation with AlexaFluor488-cadaverine and in antigen binding assays. Thus, using novel glutamine donor peptides, our technology provides an alternative enzymatic conjugation strategy for the engrafment of different payloads resulting in homogeneous batches, without unconjugated species

A cancer drug's effectiveness is contingent upon on its ability to reach all parts of the tumor. The distribution of drug in the tumor depends on several transport processes and depends on the physicochemical properties of the drug. These factors can lead to highly heterogeneous distributions of drug in the tumor interstitial space, leaving parts of the tumor unreached, and make it difficult to predict cellular exposure and understand its dependence on key system parameters. Theoretical models are powerful tools that can provide insight by simulating conditions that cannot be achieved or observed experimentally. Here, a Green's function method is utilized to simulate three-dimensional time-dependent diffusion and uptake of drugs in solid tumors with realistic vascular geometry. Regimes dependent on the time scales for transport are used to determine whether spatial and temporal effects must be resolved to predict cellular exposure. Simulations are performed to show the relationship between the plasma pharmacokinetics and cellular exposure for these regimes. Steep gradients in concentration arise when time scales for diffusion and uptake are comparable, implying that models based on well mixed compartments are inaccurate. Effects of linear and nonlinear kinetics of drug uptake on cellular exposure are demonstrated. The drug doxorubicin is commonly used against solid tumors. Cellular exposure to doxorubicin is complicated in vivo by its transport and physicochemical properties. The Green's function method is used to describe the in vivo transport and kinetics of doxorubicin, using parameters derived from in vitro results. Simulations show agreement with observed in vivo distributions of doxorubicin in tumor tissue as well as in vitro kinetics, and provide a link between the two types of experimental observations. The method is applied to the class of cancer drugs called antibody-drug conjugates (ADCs) which consist of a humanized antibody conjugated to extremely toxic small molecular weight drugs. ADCs exhibit complex in vivo kinetics dependent on many design parameters. A phenomenon exhibited by ADCs is the bystander effect, i.e. non-targeted cell killing, which is difficult to analyze based on in vivo observations. Simulations results agree with the observed in vivo distribution of ADCs in tumor tissue and with experimentally observed bystander effects. In summary, the the models presented here provide a novel approach for simulating the complex transport and cellular uptake kinetics exhibited by several cancer drugs. The models give a mechanistic basis for predicting cellular exposure to drugs which can aid, explain, and direct experimental approaches for improving cancer treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20569号 / 医科博第79号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 江藤 浩之, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Yes / The duocarmycins are potent antitumour agents with potential in the development of antibody drug conjugates (ADCs) as well as being clinical candidates in their own right. In this paper, we describe the synthesis of a duocarmycin monomer (DSA) that is suitably protected for utilisation in solid phase synthesis. The synthesis was performed on a large scale and the resulting racemic protected Fmoc-DSA subunit was separated by supercritical fluid chromatography (SFC) into the single enantiomers. Application to solid phase synthesis methodology gave a series of monomeric and extended duocarmycin analogues with amino acid substituents. The DNA sequence selectivity was similar to previous reports for both the monomeric and extended compounds. The substitution at the C-terminus of the duocarmycin caused a decrease in antiproliferative activity for all of the compounds studied. An extended compound containing an alanine at the C-terminus was converted to the primary amide or to an extended structure containing a terminal tertiary amine but this had no beneficial effects on biological activity. / MJS was funded by Novartis and UEA. We thank the EPSRC Mass Spectrometry Service, Swansea. We thank Richard Robinson and Julia Hatto at Novartis for help in the large scale synthesis.

The aplyronines are a family of 24-membered macrolides of polyketide origin, isolated from the Japanese sea hare Aplysia kurodai. They exhibit an exceptional biological activity profile, acting through an actin and tubulin dual-targeting mechanism, with subnanomolar growth inhibitory potency against a diverse range of cancer cell lines. These characteristics render the aplyronines ideal payloads for antibody-drug conjugates but their prohibitively low natural abundance calls for an efficient total synthesis to overcome the supply issue. This dissertation describes the efforts towards developing a second-generation Paterson synthesis of the macrocyclic core of the aplyronines, focused on improving the scalability and selectivity of key transformations. Chapter 1 details the isolation, biological background and previous synthetic efforts towards the aplyronines to illustrate their therapeutic potential and the challenges associated with material sourcing by chemical synthesis. Chapter 2 presents the existing body of work on the aplyronine project within the Paterson group, highlighting the lessons learned over the past two decades and shortcomings to be addressed. Chapter 3 discusses a revised protecting group strategy towards the C1-C27 macrocyclic alcohol 159 with fewer manipulation steps. A refined reaction sequence featuring titanium aldol methodology and an enzymatic desymmetrisation process delivered multigram stocks of the C15-C27 aldehyde 161 upon scale- up, testifying to the robustness of the devised route. Synthesis of the C1-C14 northern fragment 253 closely followed the existing boron aldol approach with optimisation of the C11-C12 alkylation step, geared towards enhancing the regioselectivity. Chapter 4 describes the coupling of the two major fragments using an Horner-Wadsworth-Emmons reaction to assemble the C1-C27 backbone of the cyclic aplyronine core and suitably adjusted endgame steps to enable a one-step oxidative unmasking of the macrolactonisation sites. The first-generation intermediate 159 was accessed via site-specific Yamaguchi esterification and orthogonal deprotection of the C27 allyl carbonate. Discussion in Chapter 5 includes the appendage of the C28-C34 side chain 118, prepared by the known sequence, and suggestions for the future direction of the second-generation route with the outlook of linker appendage for the purposes of antibody-drug conjugate development.

Au cours de la dernière décennie, les anticorps conjugués à des médicaments cytotoxiques ou des oligonucléotides ont acquis une grande attention dans la communauté scientifique en raison des propriétés uniques des anticorps, tels que leur long temps de circulation dans le sérum et leur sélectivité élevée par rapport à leur cible. Par exemple, les conjugués d'anticorps (ACs) sont de plus en plus appliqués en thérapie ciblée contre le cancer ou en bioimagerie. Par conséquent, le développement de méthodologies fiables pour la préparation des AC est actuellement en pleine expansion. Cependant, la conjugaison et la préparation contrôlables des ACs avec une structure définie rencontrent encore de nombreux obstacles en raison de l'excès élevé et de la variété des groupes réactifs dans la structure des anticorps, qui sont accessibles pour la conjugaison. En outre, les technologies de liaison actuelles sont basées sur la réaction de maléimide-thiol, produisant des adduits, qui sont instables dans le sang. Ce travail se concentre sur les approches chimiques pour la fonctionnalisation fiable des anticorps, qui permettent la préparation d'ACs stables présentant un ratio anticorps/principe actif bien défini. La première partie est consacrée à la conception et au développement du réactif maléimide-dioxane, solution auto-hydrolysable et stable dans le sérum, comme alternative à la chimie classique du maléimide. La deuxième partie est consacrée à l'évaluation de la réactivité sélective des différents acides aminés portés par les anticorps par spectrométrie de masse native à haute résolution. Finalement, une nouvelle technologie permettant d’obtenir des ACs stables avec un ratio anticorps/principe actif contrôlé est présentée au lecteur dans une 3ème partie. / Within the last decade, antibodies conjugated to cytotoxic drugs or oligonucleotides have gained a great attention in scientific community owing to the unique properties of the antibodies, such as their long circulation time in serum and high selectivity against their target. For instance, antibody conjugates (ACs) are increasingly applied for targeted cancer therapy or bioimaging. Consequently, the development of reliable methodologies for ACs preparation is currently of high demand. However, the controllable conjugation and preparation of ACs with defined structure are still challenging due to high excess and variety of reactive groups in antibody structure, which are accessible for conjugation. Moreover, current linker technologies are based on the maleimide-thiol reaction, yielding adducts, which are unstable during circulation in blood.This work is focused on chemical approaches for the reliable antibody functionalisation, which enable the preparation of stable ACs with well-defined payload to antibody ratios. The first part is devoted to design and development of maleimide-dioxane reagents as self-hydrolysable and serum-stable alternative to classical maleimide chemistry. The second part is dedicated to a screening approach for evaluation of residue-selective functionalities in reactions with an antibody using high resolution native mass spectrometry. Finally, in the third part the reader is introduced with a novel technology, which enables efficient preparation of stable ACs with a defined degree of conjugation and particularly mono-functionalisation of antibodies.