Current methods for the synthesis of homogeneous antibody–drug conjugates

AbstractDevelopment of efficient and safe cancer therapy is one of the major challenges of the modern medicine. Over the last few years antibody–drug conjugates (ADCs) have become a powerful tool in cancer treatment with two of them, Adcetris® (brentuximab vedotin) and Kadcyla® (ado-trastuzumab emtansine), having recently been approved by the Food and Drug Administration (FDA). Essentially, an ADC is a bioconjugate that comprises a monoclonal antibody that specifically binds tumor surface antigen and a highly potent drug, which is attached to the antibody via either cleavable or stable linker. This approach ensures specificity and efficacy in fighting cancer cells, while healthy tissues remain largely unaffected.Conventional ADCs, that employ cysteine or lysine residues as conjugation sites, are highly heterogeneous. This means that the species contain various populations of the ADCs with different drug-to-antibody ratios (DARs) and different drug load distributions. DAR and drug-load distribution are essential parameters of ADCs as they determine their stability and efficacy. Therefore, various drug-loaded forms of ADCs (usually from zero to eight conjugated molecules per antibody) may have distinct pharmacokinetics (PK) in vivo and may differ in clinical performance. Recently, a significant progress has been made in the field of site-specific conjugation which resulted in a number of strategies for synthesis of the homogeneous ADCs. This review describes newly-developed methods that ensure homogeneity of the ADCs including use of engineered reactive cysteine residues (THIOMAB), unnatural amino acids, aldehyde tags, enzymatic transglutaminase- and glycotransferase-based approaches and novel chemical methods. Furthermore, we briefly discuss the limitation of these methods emphasizing the need for further improvement in the ADC design and development

Dissecting roles and regulation of the fission yeast kinetochore protein Spc7

The accurate transmission of genetic material in the germline is of vital importance to the offspring since the entire organism is affected. It is therefore critical that chromosomes are faithfully distributed during the formation of gametes in a specialised form of cell division known as meiosis. Errors in meiotic cell divisions are a frequent cause of infertility, miscarriages, and birth defects in humans. The meiotic chromatin undergoes dynamic rearrangements during these divisions that are poorly understood at the molecular level. In prophase of the first meiotic division, the chromatin of Drosophila melanogaster oocytes detaches from the nuclear envelope to form a compact spherical cluster known as karyosome. It was previously shown that the karyosome is required for faithful chromosome segregation, but knowledge about its formation and maintenance is limited. I wish to understand how karyosome formation is regulated and identified genes important for karyosome formation in a genome-wide cytological screen of Drosophila melanogaster oocytes. The screen comprised 3,916 candidate genes expressed in ovaries, of which 209 genes showed karyosome defects upon knockdown. I found that genes encoding mitochondrial proteins, including electron transport chain components, are overrepresented amongst genes whose knockdown results in severe and reproducible karyosome defects. Interestingly, mitochondrial dysfunction induced a distinct karyosome defect characterised by three individual chromatin clusters in proximity to the nuclear envelope. Furthermore, my studies revealed that mitochondrial dysfunction not only impairs karyosome formation, but also karyosome maintenance throughout mid-oogenesis and synaptonemal complex dynamics. I asked how mitochondrial dysfunction triggers karyosome defects and aimed at further mechanistical insights. I found that mitochondrial dysfunction forces a low percentage of oocytes into apoptotic cell death, but karyosome defects occur independent of apoptosis. The knockdown of ATP synthase subunits induced the distinct karyosome defects observed upon mitochondrial dysfunction, suggesting a direct link between the karyosome phenotype and reduced levels of cellular ATP. I further determined the dependence of observed karyosome abnormalities on meiotic checkpoint activation. My work thus identified a set of genes with reproducible and checkpoint-independent karyosome defects upon knockdown. The uncharacterised function of these genes in karyosome formation and maintenance remains to be investigated and future research will pave the way for a better understanding of the karyosome at a molecular level. Furthermore, I established a link between mitochondrial dysfunction and a karyosome phenotype characterised by chromatin attachment to the nuclear envelope. The functional role of mitochondria is an increasingly important consideration in both male and female fertility. My study therefore provides a novel insight, considering that mechanistical details on how mitochondrial diseases link to infertility are sparse

Molecular architecture of the Dam1 complex-microtubule 1 interaction

Mitosis is a highly regulated process that allows the equal distribution of the genetic material to the daughter cells. Chromosome segregation requires the formation of a bipolar mitotic spindle and assembly of a multi-protein structure termed the kinetochore to mediate attachments between condensed chromosomes and spindle microtubules. In budding yeast, a single microtubule attaches to each kinetochore, necessitating robustness and processivity of this kinetochore–microtubule attachment. The yeast kinetochore-localized Dam1 complex forms a direct interaction with the spindle microtubule. In vitro, the Dam1 complex assembles as a ring around microtubules and couples microtubule depolymerization with cargo movement. However, the subunit organization within the Dam1 complex, its higher-order oligomerization and how it interacts with microtubules remain under debate. Here, we used chemical cross-linking and mass spectrometry to define the architecture and subunit organization of the Dam1 complex. This work reveals that both the C termini of Duo1 and Dam1 subunits interact with the microtubule and are critical for microtubule binding of the Dam1 complex, placing Duo1 and Dam1 on the inside of the ring structure. Integrating this information with available structural data, we provide a coherent model for how the Dam1 complex self-assembles around microtubules

Identification and characterization of aptameric inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) plays a pivotal role in innate immunity, inflammation and tissue remodelling. Aberrant proteolytic activity of HNE contributes to organ destruction in various chronic inflammatory diseases including emphysema, asthma, and cystic fibrosis. Therefore, elastase inhibitors could alleviate the progression of these disorders. Here, we used systematic evolution of ligands by exponential enrichment (SELEX) to develop single-stranded DNA aptamers that specifically target HNE. We determined the specificity of the designed inhibitors and their inhibitory efficacy against HNE using biochemical and in vitro methods, including an assay of neutrophil activity. Our aptamers inhibit the elastinolytic activity of HNE with nanomolar potency, and are highly specific for HNE and do not target other tested human proteases. As such, this study provides lead compounds suitable for the evaluation of their tissue-protective potential in animal models

Molecular platforms for targeted drug delivery

The targeted delivery of bioactive molecules to the appropriate site of action, one of the critical focuses of pharmaceutical research, improves therapeutic outcomes and increases safety at the same time; a concept envisaged by Ehrlich over 100 years ago when he described the "magic bullet" model. In the following decades, a considerable amount of research effort combined with enormous investment has carried selective drug targeting into clinical practice via the advent of monoclonal antibodies (mAbs) and antibody-drug conjugates derivatives. Additionally, a deeper understanding of physiopathological conditions of disease has permitted the tailored design of targeted drug delivery platforms that carry drugs, many copies of the same drug, and different drugs in combination to the appropriate site of action least selectively or preferentially. The acquired know-how has provided the field with the design rationale to develop a successful delivery system that will provide new and improved means to treat many intractable diseases and disorders. In this review, we discuss a wide range of molecular platforms for drug delivery, and focus on those with more success in the clinic, given their potential for targeted therapies

The role of language in faith and pastoral ministry

Pół wieku od zakończenia Vaticanum II (1962-1965) często zapomina się o dobrodziejstwie, jakie przyniósł sobór całemu Kościołowi w czynnym przeżywaniu wiary, liturgii przez wiernych. A stało się to możliwe przez dopuszczenie języków narodowych do liturgii. Autor niniejszego artykułu na przykładzie języka romani pokrótce ukazuje, jaką rolę pełni i powinien pełnić język ojczysty, w tym przypadku romski, w wierze i duszpasterstwie dla tych osób, zarówno duchownych, jak i wiernych świeckich, które się nim posługują i w ten sposób oddają cześć Bogu w duchu i w prawdzie. Rola, jaką odgrywa język ojczysty w wierze i duszpasterstwie, winna wpływać na troskę duszpasterzy o jego używanie, m.in. w posłudze słowa, szczególnie w czasie sprawowania sakramentów św. i nabożeństw liturgicznych, aby ukazać współczesnemu człowiekowi prawdę, piękno i radość przeżywanej przez niego wiary, by była ona żywa.It has been half of a century since the end of Vatican Council II (1962-1965) and it is often forgotten about the benevolence of the Council brought to the whole Church and made the life of faith and liturgy more active for believers. It was possibile by allowing to use national languages in the liturgy. The author of this article shows, on the example of Romany language, what is the role of a native language in faith and pastoral care for people, both clergy and laity, who speak this language and worship God in spirit and truth. The role of the mother tongue in faith and pastoral ministry should influence pastors in its usage for example in the ministry of the word, especially in performing the sacraments and liturgical celebrations. It is all to show to the modern man the truth, beauty, and joy experienced by the faith and makes it alive

Retable from Lubowo (Łubowo). An example of late gothic sculpture and the lay piety of the and of 15th and the beginning of 16th century in Western Pomerania

Ołtarz z Łubowa pojawił się w ostatnich miesiącach II wojny światowej. W czasie cofania linii frontu niemiecki oficer zdeponował paczkę u rodziny ze Starowic. Po wojnie, gdy pakunek został rozpieczętowany, depozytariusze odkryli w niej późnośredniowieczny tryptyk. Został on umieszczony w kościele filialnym w Starowicach, a w 1975 roku w kościele parafialnym w Łubowie. Ołtarz został odrestaurowany w latach 1974–1975 oraz w latach 1994–1995 przez pracownie konserwatorskie w Gdańsku i Szczecinie. W centralnej części retabulum znajdują się figury Matki Bożej z Dzieciątkiem i dwóch świętych: Jakuba Starszego i Antoniego Opata. W dwóch skrzydłach znajdują się kwatery z postaciami świętych. Lewe skrzydło zawiera figury świętych Jana Chrzciciela, Piotra i Pawła powyżej oraz Barbary, Katarzyny i Dionizego poniżej. W prawym skrzydle występuje przedstawienie dwóch niezidentyfikowanych świętych oraz Jana Ewangelisty w górnej kwaterze, a w dolnej Małgorzaty, Doroty i Wawrzyńca. Ideą przewodnią obiektu jest historia Odkupienia reprezentowana przez Madonnę z Dzieciątkiem, które w rączkach ma owoc – jabłko, symbol grzechów i ich odkupienia. Grupy postaci reprezentują poszczególne kategorie świętych – apostołów, męczenników, dziewic i spowiedników. Ołtarz powstał w kontekście życia późnogotyckich społeczności miejskich Pomorza – miast, które zajmowały się handlem międzynarodowym – jako szczególne narzędzie komunikacji wizualnej głoszące orędzie zbawienia. Zofia Krzymuska-Fafius dostrzegła w nim analogie stylistyczne nawiązujące do niemieckich realizacji późnogotyckich w Osnabrück, Bordesholm i Würzburgu, a także powiązała go ze środowiskiem ołtarza pasyjnego ze Szczecina Dąbia. Jednak jednocześnie istnieje możliwość zestawienia go z najbliższymi ołtarzami ze Starego Ludzicka i Koszalina jako przykładami podobnych koncepcji późnogotyckich retabulów.The altarpiece of Lubowo (Łubowo) appeared in the time of the last months of Second World War. In fact, a German officer deposed the parcel by the Pomeranian family. After the war, when the parcel was unpacked, the depositors discovered the late medieval triptych on. It has been conceded to the filial catholic church in Starowice and in 1975 placed in the parish church in Lubowo (Łubowo). The altarpiece has been restored in the years 1974–1975 and in the years 1994–1995 by the renovation ateliers in Gdansk (Gdańsk) and Szczecin. The retable has a central part with the figures of Mother of God with Child and two saints – James the Older and Antony Abbot. The two wings contain the quarters with figures of saint. The left wing contains the figures of saints John the Baptiste, Peter and Paul above and Barbara, Catharina and Dion¬isius below. The right wing contains the representations of two unidentified saint and saint John the Evangelist above and Margaretha, Dorothy and Laurent below. The message of this composition of figures consist on the Redemption represented in the figure of Madonna with Child, because the little Jesus bears the apple, symbol of sins and its redemption. The groups of figures represents the particular categories of Saints – apostles matyrs, virgins, confessors. The altarpiece has been created in the context of life of urban communities of Pomerania – the cities who managed the international commerce. The citizens needed a particular tool of visual communication, who proclaimed the Christian realities, Zofia Krzymuska-Fafius recognized the stylistic analogies which connect our altarpiece with different other realizations in the German areas. She founded similarities in the late gothic works of sculpture from Szczecin Dabie (Dąbie), Osnabrück, Bordesholm and Würzburg. But it is possible to compare our retable with different example of Pomeranian altarpiece from Stary Ludzick and Koszalin