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Multivalent Functionalized Dextran-Antibody Conjugates for Efficient Tumor Cell Killing

Schneider, Hendrik Peter Günter (2020)
Multivalent Functionalized Dextran-Antibody Conjugates for Efficient Tumor Cell Killing.
Technische Universität Darmstadt
doi: 10.25534/tuprints-00011502
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

Despite impressive progress in medical care and extensive investigation in the field of human malignancies, cancer still represents a major global health issue, and triggering apoptosis of tumor cells has been in focus of cancer research for decades. In addition to classical attempts like surgery, radiotherapy, and chemotherapy, a number of novel methodologies have recently fallen in the limelight, with monoclonal antibodies being the most prominent actors at the therapeutic scene. With their introduction, the so-called targeted therapy has finally become achievable for the treatment of malignant tumors. Having evolved from Paul Ehrlich’s “Magic Bullet” concept that described directing a toxic compound exclusively to a disease-causing organism, antibody-drug conjugates (ADCs) were developed. This class of compounds is aimed at site-selective delivery of cytotoxic agents to target cells expressing a cancer-related antigen. Combining the targeting properties of an antibody with the killing properties of a potent cytotoxin, these constructs were applied in countless approaches intending to treat tumor patients. To date, six ADCs have been marketed and over 60 ones are currently in clinical trials. However, several issues still require improvement, among them toxicity, efficacy and pharmacokinetics.

The first investigation in the context of the present cumulative study was focused on the generation of highly hydrophilic ADCs characterized by a high drug-to-antibody ratio (DAR). However, since only a limited number of the administered ADCs is reported to actually reach their cellular target, either highly potent toxins or a higher number of the less-potent ones are prerequisites of these compounds to reach efficacy. Considering the hydrophobic character of most commonly applied potent cytotoxins, these conjugates often suffer from poor hydrophilicity depending on the addressed conjugation site as well as the number and character of the applied toxin units. Further, hydrophobicity of ADCs was reported to raise problems due to aggregation and recognizing by multidrug resistance (MDR) transporters, thus the number, site, and hydrophobicity of the conjugated toxin strongly influences stability, pharmacokinetic properties and the efficacy of ADCs. In this work we addressed these issues simultaneously by designing a novel class of hybrid ADCs combining ability to balance and even recompense the hydrophobicity of commonly applied highly hydrophobic cytotoxins with an option for the attachment of multiple payloads, which may further enable the application of less potent, thus less harmful for the healthy tissues, cytotoxins. To that end, we applied dextran, an FDA-approved polysaccharide, consisting mainly of α-1,6-linked oligo-D-glucose units as multivalency-generating modular scaffold for payload attachment. This glucan, reported to enhance half-life, to improve thermal stability and pharmacokinetic properties, and to reduce immunogenicity of conjugated proteins, opens certain space for chemical modifications, namely conjugation of a) a desired number of payloads to the repeating glucose units at the respective hydroxy groups, and b) at the reducing end that comprises an orthogonally addressable aldehyde. In this work, a strategy to combine an enzyme-catalyzed site-specific conjugation of the dextran scaffold, equipped with multiple reactive moieties for payload loading, to antibodies was developed, resulting in promising constructs for the generation of high-DAR ADCs. Synthetically, a combination of reductive amination of dextran’s reducing end with a protected diamine followed by site-selective carboxyethylation at the C2-position of the glucose repeating units led to dextran bearing dually addressable reactive moieties. Notably, our synthetic procedure allowed adjustment of the number of addressable sites at the repeating glucose monomers for toxin conjugation to the desired amount of copies. Subsequent conjugation of azide-bearing aliphatic amines at the repeating units upon amidation gave a scaffold comprising multiply addressable “click” sites. Demasking of the protected amine at the reducing end yielded a multivalent scaffold combining multiple and solitaire orthogonal addressable sites. Thus, at its amine site it could be easily conjugated to the protein of interest – in this particular case, to the therapeutic antibody trastuzumab – via enzymatic catalysis by microbial transglutaminase (mTG). For this purpose, trastuzumab was engineered to possess an adequate mTG recognition motif at the C-terminus of the heavy chain. The azides at the sugar monomers remained for the decoration with monomethyl auristatin E (MMAE) – a highly toxic and extraordinary hydrophobic compound. To conclude, for the first-time dextran was site-specifically conjugated to a functional antibody via its reducing end, leaving the polysaccharide backbone intact and subsequently equipped with multiple MMAEs by strain-promoted azide-alkyne cycloaddition (SPAAC) in a desired number of copies without corrupting the polysaccharide backbone. These hybrid constructs, called dextramabs, were found not only readily soluble in aqueous buffers, but at least as hydrophilic as the parental antibody trastuzumab, even when conjugated with eleven highly hydrophobic MMAE counterparts. The binding properties of all generated constructs were not affected, as demonstrated by comparable KD values on HER2-positive SK-BR-3 cells (unmodified trastuzumab: KD = 4.9 nM, dextramab (DAR 8): KD = 5.9 nM). Our synthetic dextramabs showed potent subnanomolar cytotoxicity (IC50 = 100 pM) in cell proliferation assays on HER2-positive SK-BR-3 breast cancer cells and no cytotoxicity on HER2-negative control cells in vitro. These site-specifically assembled ADCs may combine the beneficial pharmacokinetic properties, as their protein counterparts are loaded with dextran, therefore possess higher hydrodynamic radius, with the possibility to attach a tailored number of payloads. Generally, our concept represents a promising approach for the generation of highly hydrophilic site-specific ADCs characterized by a high DAR. Follow-up animal studies will unveil if dextramabs hold promise for the novel class of ADCs with high potency, low immunogenicity and enhanced in vivo half-life.

In the second investigation we studied applicability of dextran polysaccharide scaffold as carrier for apoptosis-triggering payloads of diverse nature, which act by addressing distinct intra- or extracellular targets. Hence, additionally to the above-mentioned high-DAR ADCs we were focused at validating dextran as a platform for multimerization of cancer-relevant ligands. First, death receptor 5 (DR5) was chosen as a model target expressed on the cell surface. As it is activated by oligomerization/aggregation, we aimed at constructing a flexible scaffold able to bypass the reported need for spatial ligand orientation for efficient DR5-mediated cellular cytotoxicity. Thus, we designed a molecular architecture comprising a polysaccharide scaffold carrying the desired number of DR5 peptidic binders able to efficiently trigger apoptosis upon DR5 receptor clustering. Herein, apoptosis of cancer cells was mediated by multivalent binding to and clustering of a receptor located on the cell surface, which is in fact contrary to the first approach investigated in this work, which relied on a high number of a very potent cytotoxin that only upon internalization inhibits cell division by blocking the polymerization of tubulin. Thus, dextran was loaded with on average 11 or 13.4 peptidic binders, namely death receptor 5 targeting peptides (DR5TP). The resulting constructs were found potent apoptosis-inducing conjugates possessing double-digit nanomolar half-maximal effective concentration (EC50) values on DR5-positive COLO205 colon cells and Jurkat T lymphocytes in vitro. Moreover, conjugation to glutamine 295 of an aglycosylated fragment crystallizable (Fc) fragment of a monoclonal antibody (mAb) by site-specific mTG-catalyzed conjugation resulted in constructs that showed selective DR5 binding upon flow cytometric analysis and further did not impair the potency of the generated multivalent scaffolds. In contrast, these protein-polysaccharide-peptide hybrids demonstrated higher potency in vitro (EC50 = 1.9 - 6.7 nM). Notably, in this approach binding is not mediated by the protein, but rather by the ligand-bearing dextran counterpart. Thus, addition of a second targeting moiety, e.g. application of a full-length antibody, would be an interesting prospective study that opens the possibility of bispecific targeting, which may result in enhanced safety and efficacy. Furthermore, the generated DR5TP-dextran and the Fc-bound counterparts were able to circumvent the mentioned need for spatial orientation of ligands due to additional flexibility provided by dextran scaffold. Our study further underlines the modularity of dextran as carrier for different payloads addressing various targets. In addition, this approach may help overcoming the reported off-target toxicity for multimeric high-affinity protein-based constructs, e.g. TAS266, by the application of low-affinity peptidic binders. This may lead to better tolerability in vivo, conditioned by lower retention on healthy cells expressing minor levels of DR5, which might be additionally improved by prospective bispecific targeting.

The third part of this work was aimed at relieving the current lack of satisfactory overviews on mTG-mediated generation of homogeneous ADCs. Since most reviews dealing with ADCs cover a broad scope of topics, but usually very briefly, an in-depth comparative survey was highly required. However, a detailed overview of the factors influencing the resulting architectures in view of stability, potency, efficiency, etc. was still missing. A comprehensive summary of the reported strategies may enable tailoring of existing methods for mTG-promoted conjugation to the needs of particular research projects. On these grounds, the originated review was intended not only to enumerate the applied approaches for site-specific conjugation with respect to ADC assembly, but to map out the research groups and companies working on mTG-mediated generation of antibody-drug conjugates. Our review, gives a thorough overview of conjugation methodologies, addressed conjugation motifs or sites, applied cellular targets, linkers, and cytotoxic cargoes. Thus, it highlights pioneering routes and techniques, recent progress and remaining limitations of mTG-assisted assembly of ADCs.

Furthermore, a study aimed at assessment of possibilities offered by dextran as a multivalency-promoting framework was performed. In a preliminary proof-of-concept study dextran was applied as a vehicle for multiple attachment of metal-chelating agents able to carrier valuable ions for radio-imaging or -therapy. Thus, dextran polymer was equipped with a desired number of a widely applied metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The assembled DOTA-dextran conjugates were able to carry 3.2 or 5,3 metal ions per polysaccharide chain, respectively, as shown by photometric analysis of the formed complexes with Cu2+. In vivo biodistribution studies in mice are currently ongoing. This proof-of-concept study should answer the question, whether these novel molecular hybrids are suitable for in vivo applications and whether conjugation to commonly applied binders (affibodies) that suffer from hydrophobicity and in consequence poor solubility, aggregation and precipitation, results in beneficial properties. This approach further strengthens the presumption that dextran represents a promising modular scaffold for multivalent attachment and tailoring of diverse payloads.

Typ des Eintrags: Dissertation
Erschienen: 2020
Autor(en): Schneider, Hendrik Peter Günter
Art des Eintrags: Erstveröffentlichung
Titel: Multivalent Functionalized Dextran-Antibody Conjugates for Efficient Tumor Cell Killing
Sprache: Englisch
Referenten: Kolmar, Prof. Dr. Harald ; Schmitz, Prof. Dr. Katja
Publikationsjahr: 2020
Ort: Darmstadt
Datum der mündlichen Prüfung: 10 Februar 2020
DOI: 10.25534/tuprints-00011502
URL / URN: https://tuprints.ulb.tu-darmstadt.de/11502
Kurzbeschreibung (Abstract):

Despite impressive progress in medical care and extensive investigation in the field of human malignancies, cancer still represents a major global health issue, and triggering apoptosis of tumor cells has been in focus of cancer research for decades. In addition to classical attempts like surgery, radiotherapy, and chemotherapy, a number of novel methodologies have recently fallen in the limelight, with monoclonal antibodies being the most prominent actors at the therapeutic scene. With their introduction, the so-called targeted therapy has finally become achievable for the treatment of malignant tumors. Having evolved from Paul Ehrlich’s “Magic Bullet” concept that described directing a toxic compound exclusively to a disease-causing organism, antibody-drug conjugates (ADCs) were developed. This class of compounds is aimed at site-selective delivery of cytotoxic agents to target cells expressing a cancer-related antigen. Combining the targeting properties of an antibody with the killing properties of a potent cytotoxin, these constructs were applied in countless approaches intending to treat tumor patients. To date, six ADCs have been marketed and over 60 ones are currently in clinical trials. However, several issues still require improvement, among them toxicity, efficacy and pharmacokinetics.

The first investigation in the context of the present cumulative study was focused on the generation of highly hydrophilic ADCs characterized by a high drug-to-antibody ratio (DAR). However, since only a limited number of the administered ADCs is reported to actually reach their cellular target, either highly potent toxins or a higher number of the less-potent ones are prerequisites of these compounds to reach efficacy. Considering the hydrophobic character of most commonly applied potent cytotoxins, these conjugates often suffer from poor hydrophilicity depending on the addressed conjugation site as well as the number and character of the applied toxin units. Further, hydrophobicity of ADCs was reported to raise problems due to aggregation and recognizing by multidrug resistance (MDR) transporters, thus the number, site, and hydrophobicity of the conjugated toxin strongly influences stability, pharmacokinetic properties and the efficacy of ADCs. In this work we addressed these issues simultaneously by designing a novel class of hybrid ADCs combining ability to balance and even recompense the hydrophobicity of commonly applied highly hydrophobic cytotoxins with an option for the attachment of multiple payloads, which may further enable the application of less potent, thus less harmful for the healthy tissues, cytotoxins. To that end, we applied dextran, an FDA-approved polysaccharide, consisting mainly of α-1,6-linked oligo-D-glucose units as multivalency-generating modular scaffold for payload attachment. This glucan, reported to enhance half-life, to improve thermal stability and pharmacokinetic properties, and to reduce immunogenicity of conjugated proteins, opens certain space for chemical modifications, namely conjugation of a) a desired number of payloads to the repeating glucose units at the respective hydroxy groups, and b) at the reducing end that comprises an orthogonally addressable aldehyde. In this work, a strategy to combine an enzyme-catalyzed site-specific conjugation of the dextran scaffold, equipped with multiple reactive moieties for payload loading, to antibodies was developed, resulting in promising constructs for the generation of high-DAR ADCs. Synthetically, a combination of reductive amination of dextran’s reducing end with a protected diamine followed by site-selective carboxyethylation at the C2-position of the glucose repeating units led to dextran bearing dually addressable reactive moieties. Notably, our synthetic procedure allowed adjustment of the number of addressable sites at the repeating glucose monomers for toxin conjugation to the desired amount of copies. Subsequent conjugation of azide-bearing aliphatic amines at the repeating units upon amidation gave a scaffold comprising multiply addressable “click” sites. Demasking of the protected amine at the reducing end yielded a multivalent scaffold combining multiple and solitaire orthogonal addressable sites. Thus, at its amine site it could be easily conjugated to the protein of interest – in this particular case, to the therapeutic antibody trastuzumab – via enzymatic catalysis by microbial transglutaminase (mTG). For this purpose, trastuzumab was engineered to possess an adequate mTG recognition motif at the C-terminus of the heavy chain. The azides at the sugar monomers remained for the decoration with monomethyl auristatin E (MMAE) – a highly toxic and extraordinary hydrophobic compound. To conclude, for the first-time dextran was site-specifically conjugated to a functional antibody via its reducing end, leaving the polysaccharide backbone intact and subsequently equipped with multiple MMAEs by strain-promoted azide-alkyne cycloaddition (SPAAC) in a desired number of copies without corrupting the polysaccharide backbone. These hybrid constructs, called dextramabs, were found not only readily soluble in aqueous buffers, but at least as hydrophilic as the parental antibody trastuzumab, even when conjugated with eleven highly hydrophobic MMAE counterparts. The binding properties of all generated constructs were not affected, as demonstrated by comparable KD values on HER2-positive SK-BR-3 cells (unmodified trastuzumab: KD = 4.9 nM, dextramab (DAR 8): KD = 5.9 nM). Our synthetic dextramabs showed potent subnanomolar cytotoxicity (IC50 = 100 pM) in cell proliferation assays on HER2-positive SK-BR-3 breast cancer cells and no cytotoxicity on HER2-negative control cells in vitro. These site-specifically assembled ADCs may combine the beneficial pharmacokinetic properties, as their protein counterparts are loaded with dextran, therefore possess higher hydrodynamic radius, with the possibility to attach a tailored number of payloads. Generally, our concept represents a promising approach for the generation of highly hydrophilic site-specific ADCs characterized by a high DAR. Follow-up animal studies will unveil if dextramabs hold promise for the novel class of ADCs with high potency, low immunogenicity and enhanced in vivo half-life.

In the second investigation we studied applicability of dextran polysaccharide scaffold as carrier for apoptosis-triggering payloads of diverse nature, which act by addressing distinct intra- or extracellular targets. Hence, additionally to the above-mentioned high-DAR ADCs we were focused at validating dextran as a platform for multimerization of cancer-relevant ligands. First, death receptor 5 (DR5) was chosen as a model target expressed on the cell surface. As it is activated by oligomerization/aggregation, we aimed at constructing a flexible scaffold able to bypass the reported need for spatial ligand orientation for efficient DR5-mediated cellular cytotoxicity. Thus, we designed a molecular architecture comprising a polysaccharide scaffold carrying the desired number of DR5 peptidic binders able to efficiently trigger apoptosis upon DR5 receptor clustering. Herein, apoptosis of cancer cells was mediated by multivalent binding to and clustering of a receptor located on the cell surface, which is in fact contrary to the first approach investigated in this work, which relied on a high number of a very potent cytotoxin that only upon internalization inhibits cell division by blocking the polymerization of tubulin. Thus, dextran was loaded with on average 11 or 13.4 peptidic binders, namely death receptor 5 targeting peptides (DR5TP). The resulting constructs were found potent apoptosis-inducing conjugates possessing double-digit nanomolar half-maximal effective concentration (EC50) values on DR5-positive COLO205 colon cells and Jurkat T lymphocytes in vitro. Moreover, conjugation to glutamine 295 of an aglycosylated fragment crystallizable (Fc) fragment of a monoclonal antibody (mAb) by site-specific mTG-catalyzed conjugation resulted in constructs that showed selective DR5 binding upon flow cytometric analysis and further did not impair the potency of the generated multivalent scaffolds. In contrast, these protein-polysaccharide-peptide hybrids demonstrated higher potency in vitro (EC50 = 1.9 - 6.7 nM). Notably, in this approach binding is not mediated by the protein, but rather by the ligand-bearing dextran counterpart. Thus, addition of a second targeting moiety, e.g. application of a full-length antibody, would be an interesting prospective study that opens the possibility of bispecific targeting, which may result in enhanced safety and efficacy. Furthermore, the generated DR5TP-dextran and the Fc-bound counterparts were able to circumvent the mentioned need for spatial orientation of ligands due to additional flexibility provided by dextran scaffold. Our study further underlines the modularity of dextran as carrier for different payloads addressing various targets. In addition, this approach may help overcoming the reported off-target toxicity for multimeric high-affinity protein-based constructs, e.g. TAS266, by the application of low-affinity peptidic binders. This may lead to better tolerability in vivo, conditioned by lower retention on healthy cells expressing minor levels of DR5, which might be additionally improved by prospective bispecific targeting.

The third part of this work was aimed at relieving the current lack of satisfactory overviews on mTG-mediated generation of homogeneous ADCs. Since most reviews dealing with ADCs cover a broad scope of topics, but usually very briefly, an in-depth comparative survey was highly required. However, a detailed overview of the factors influencing the resulting architectures in view of stability, potency, efficiency, etc. was still missing. A comprehensive summary of the reported strategies may enable tailoring of existing methods for mTG-promoted conjugation to the needs of particular research projects. On these grounds, the originated review was intended not only to enumerate the applied approaches for site-specific conjugation with respect to ADC assembly, but to map out the research groups and companies working on mTG-mediated generation of antibody-drug conjugates. Our review, gives a thorough overview of conjugation methodologies, addressed conjugation motifs or sites, applied cellular targets, linkers, and cytotoxic cargoes. Thus, it highlights pioneering routes and techniques, recent progress and remaining limitations of mTG-assisted assembly of ADCs.

Furthermore, a study aimed at assessment of possibilities offered by dextran as a multivalency-promoting framework was performed. In a preliminary proof-of-concept study dextran was applied as a vehicle for multiple attachment of metal-chelating agents able to carrier valuable ions for radio-imaging or -therapy. Thus, dextran polymer was equipped with a desired number of a widely applied metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The assembled DOTA-dextran conjugates were able to carry 3.2 or 5,3 metal ions per polysaccharide chain, respectively, as shown by photometric analysis of the formed complexes with Cu2+. In vivo biodistribution studies in mice are currently ongoing. This proof-of-concept study should answer the question, whether these novel molecular hybrids are suitable for in vivo applications and whether conjugation to commonly applied binders (affibodies) that suffer from hydrophobicity and in consequence poor solubility, aggregation and precipitation, results in beneficial properties. This approach further strengthens the presumption that dextran represents a promising modular scaffold for multivalent attachment and tailoring of diverse payloads.

Alternatives oder übersetztes Abstract:
Alternatives AbstractSprache

Trotz massiven medizinischen Fortschritts und ausgiebigen Untersuchungen in der Krebsforschung repräsentieren maligne Tumore immer noch eines der größten globalen Gesundheitsprobleme. Daher liegt das Auslösen der Apoptose von Tumorzellen seit Jahren im Fokus der Forschung. Neben den klassischen Ansätzen, wie Operationen, Strahlen- und Radiotherapie, sowie Chemotherapie wurden neue Methoden zur Behandlung von Krebs entwickelt. Hierzu zählt das Konzept der zielgerichteten Therapie, für das monoklonale Antikörper ein prominentes Beispiel darstellen. Eine zusätzliche Komponente dieses Ansatzes stellen Antikörper-Wirkstoff-Konjugate (ADCs) dar, die dem von Paul Ehrlich geprägten Konzept der „Zauberkugel“ entsprechen. Diese Klasse von Verbindungen zielen auf den ortspezifischen Transport und die Abgabe eines Zytostatikums an Zielzellen ab, die tumorspezifische Antigene exprimieren. Diese Konstrukte kombinieren die Bindungseigenschaften eines Antikörpers mit der Toxizität eines Zytostatikums und wurden in zahlreichen Ansätzen für die Krebstherapie eingesetzt. So wurden bis heute sechs ADCs kommerziell vermarktet und über 60 werden aktuell in klinischen Studien untersucht. Trotzdem besteht weiterhin ein gewisses Verbesserungspotential dieser potenten Verbindungen hinsichtlich ihrer Toxizität, Effizienz und pharmakokinetischen Eigenschaften.

Die erste Untersuchung im Rahmen der hier vorliegenden, kumulativen Dissertation fokussierte sich auf die Generierung von ADCs, die ein hohes Wirkstoff-zu-Antikörper Verhältnis (DAR) aufweisen und deren Hydrophilie durch Anfügen von Zytotoxinen nicht beeinträchtigt wird. Da lediglich ein geringer Anteil des verabreichten ADCs tatsächlich die adressierten Zielzellen erreicht, sind entweder hochwirksame oder eine hohe Anzahl weniger potenter Zytotoxine unerlässlich, um die gewünschte Effektivität zu erzielen. In Anbetracht des hydrophoben Charakters der am häufigsten eingesetzten Zytotoxine weisen diese Konjugate jedoch häufig eine niedrige Hydrophilie auf, welche abhängig von der adressierten Konjugationsstelle, der Anzahl und dem Charakter der angewendeten Toxineinheiten ist. Folglich hat die Hydrophobizität der konjugierten Zytostatika einen großen Einfluss auf die Stabilität sowie die pharmakokinetischen Eigenschaften und bestimmt somit die Effizienz des ADCs. So wurde beispielsweise von Problemen bezüglich Aggregation, Erkennung durch multidrug resistance (MDR) Rezeptoren und erhöhter Clearance berichtet. In dieser Arbeit sollten diese Probleme durch das Design einer neuen Klasse von Hybrid-ADCs gelöst werden, die die Hydrophobizität der im Kontext von ADCs weithin genutzten Zytostatika mindern oder sogar gänzlich kompensieren kann und zusätzlich die Möglichkeit eröffnet, eine gewünschte Menge dieser potenten Toxine pro Antikörper zur Verfügung zu stellen. Zusätzlich sollte eine zukünftige Beladung mit weniger wirksamen und folglich weniger schädlichen Toxinen ermöglicht werden, bei denen die reduzierte Toxizität durch eine erhöhte Kopienzahl kompensiert wird. Dextran, ein von der FDA zugelassenes Polysaccharid, das aus α-1,6-verknüpften Oligoglukoseeinheiten besteht, wurde in dieser Arbeit als modulares, multivalenzgenerierendes Grundgerüst für die ortsspezifische Konjugation an Antikörper und für die Beladung mit einer gewünschten Anzahl an Zytostatika verwendet. Für die Konjugation dieses Glucans an Proteine wurde berichtet, dass sie die Halbwertszeit, die thermische Stabilität sowie die pharmakokinetischen Eigenschaften dieser verbessert und deren Immunogenität verringert. Weiterhin bietet sich die Möglichkeit, chemische Modifikationen an unterschiedlichen Stellen dieses Zuckermoleküls, zur Einführung orthogonal adressierbarer Gruppen durchzuführen. So kann sowohl eine gewünschte Anzahl an Zytostatika an den Hydroxygruppen der Glukose-Wiederholungseinheiten als auch ein Erkennungsmotiv für die enzymatische Konjugation an ein Protein an das orthogonal adressierbare Aldehyd am reduzierenden Ende angebracht werden. In dieser Arbeit wurde zudem eine Synthesestrategie entwickelt, die es ermöglicht, Dextran, beladen mit einer gewünschten Anzahl orthogonal adressierbarer chemischer Gruppen zur Toxinkonjugation, mittels Enzymkatalyse ortsspezifisch an einen Antikörper zu konjugieren und anschließend mit Toxinen auszustatten. Diese Hybridverbindung ebnete den Weg zur Generierung neuer, vielversprechender hoch hydrophiler ADCs mit hohem Wirkstoff-zu-Antikörper Verhältnis. Synthetisch wurde hierfür eine reduktive Aminierung mit einem geschützten Diamin am reduzierenden Ende mit einer ortsspezifischen Carboxyethylierung an der C2-Position der Glukose-Wiederholungseinheiten verbunden, woraus ein Dextran mit zwei unterschiedlich adressierbaren reaktiven Gruppen resultierte. Dabei konnte eine gewünschte Anzahl der Carboxygruppen an den Glukose-Wiederholungseinheiten eingestellt werden. Anschließend wurden amintragende Azidlinker unter Bildung von Amidbindungen kovalent an den eingebauten Carboxygruppen der Glukose-Wiederholungseinheiten eingeführt, woraus ein Dextranrückgrat mit multiplen Azid-Funktionalitäten resultierte. Die Demaskierung des geschützten Amins am reduktiven Ende ergab schließlich dual adressierbare Polysaccharidgerüste mit multiplen „click“-baren Gruppen zur Konjugation der Zytostatika und einem primären Amin, mittels dessen eine enzymkatalysierte Konjugation an den Antikörper realisiert werden konnte. Trastuzumab, welcher genetisch mit einer Transglutaminase-Erkennungssequenz versehen wurde, diente hierbei als Modellantikörper. Die Konjugation des azidtragenden Dextrangerüsts an Trastuzumab unter Katalyse von mikrobieller Transglutaminase und die darauffolgende Konjugation eines potenten, sehr hydrophoben Zytoskelettinhibitors (Monomethylauristatin E (MMAE)), bildete ein neues Hybridkonstrukt. So wurde Dextran in dieser Arbeit zum ersten Mal ortsspezifisch an einen Antikörper gekoppelt und sukzessive mit einer gewünschten Anzahl an Zytostatika durch spannungskatalysierte Azid‐Alkin‐Cycloaddition (SPAAC) beladen. Diese neuen, als Dextramabs bezeichneten Hybridkonstrukte erwiesen sich nicht nur als gut löslich in wässrigen Puffern, sondern auch als mindestens ebenso hydrophil wie der nicht modifizierte Trastuzumab, selbst wenn das Polysaccharid mit elf hydrophoben MMAEs beladen wurde. Weiterhin zeigten die generierten Konstrukte unveränderte Bindungseigenschaften und keinen Stabilitätsverlust (Trastuzumab: KD = 4.9 nM, Dextramab (DAR 8): KD = 5.9 nM). In Zellviabilitätstests zeigten die entwickelten Dextramabs potente, subnanomolare mittlere inhibitorische Konzentration (IC50 = 100 pM) auf HER2-positiven SK-BR-3 Brustkrebszellen in vitro, wohingegen keine Toxizität für HER2-negative Kontrollzellen gefunden wurde. Dextramabs stellen eine neue Strategie für die Generierung ortsspezifisch konjugierter hoch-hydrophiler ADCs mit einem hohen DAR dar. Tierstudien werden zeigen, ob diese neue Klasse von ADCs ein vielversprechendes Konzept zur Generierung von ADCs mit hoher Wirksamkeit, geringer Immunogenität und erhöhter in vivo-Halbwertszeit repräsentiert.

In der zweiten Untersuchung wurde die Modularität von Dextran als Träger für Apoptose-induzierende Moleküle, die sowohl intrazelluläre als auch Ziele auf der Zelloberfläche adressieren, untersucht. Im Zuge dessen wurde, im Gegensatz zum ersten Ansatz, Dextran als eine Plattform zur Multimerisierung von krebsspezifischen Liganden untersucht. Der extrazellulär exprimierte Death Rezeptor 5 (DR5) wurde hierfür als Modellziel gewählt, welcher durch Aggregation beziehungsweise Multimerisierung aktiviert wird und eine Apoptose-induzierende Signal-Kaskade auslöst (death-inducing singal cascade, DISC). In dieser Arbeit wurde Dextran als Grundgerüst mit mehreren peptidischen Liganden (death receptor 5-targeting peptide, DR5TP) von DR5 bestückt und im Hinblick auf seine Apoptose-induzierende Wirkung untersucht. Hierbei sollte, im Gegensatz zu der ersten Untersuchung, der Zelltod durch Multimerisierung von Rezeptoren auf der Zelloberfläche induziert werden und nicht durch rezeptorvermittelte, endosomatische Aufnahme eines an den Antikörper gebundenen Zytostatikums. Hierfür wurde Dextran mit 11 respektive 13.4 DR5TP-Liganden beladen und auf seine Apoptose-induzierende Wirkung untersucht. Die resultierenden multivalenten Dextrangerüste zeigten mit zweistellig nanomolaren EC50-Werten (halbmaximalen effektiven Konzentrationen) auf DR5-positiven COLO205-Kolonzellen und Jurkat T-Lymphozyten eine potente Apoptose-induzierende Wirkung in vitro. Eine anschließende ortsspezifische Konjugation an Glutamin 295 eines aglykosylierten kristallisierbaren Fragments (Fragment, crystallizable, Fc) mittels mikrobieller Transglutaminase resultierte in Protein-Polysaccharid-Konjugaten, die eine selektive DR5-Bindung in durchflusszytometrischer Analyse zeigten und die im Vergleich zu den nicht an ein Protein konjugierten DR5TP-Dextranen sogar niedrigere EC50-Werte aufwiesen (EC50 = 1.9 - 6.7 nM). Interessanterweise wurde die Bindung dieser Konjugate nicht durch den Antikörper, sondern durch das mit Liganden bestückte Dextran hervorgerufen. Dies ermöglicht die zukünftige Verwendung einer zweiten Bindungseinheit im Hinblick auf bispezifisches Targeting, beispielsweise durch die Konjugation an einen Vollängen Antikörper, wodurch eventuell eine höhere Sicherheit und Effizienz realisiert werden könnte. Weiterhin ermöglichten die hergestellten Konjugate, sowohl solitär als auch gebunden an das Fc-Fragment, durch die erhöhte Flexibilität des Dextranrückgrats die beschriebene distinkte räumliche Anordnung als unumgängliche Voraussetzung für effizientes Induzieren von Apoptose zu kompensieren. Dieser Ansatz untermauert die Modularität von Dextran als Träger für unterschiedlichste Beladungen, die verschiedene Zielstrukturen zur gezielten Auslösung von Apoptose von Tumorzellen adressieren. Zukünftig könnte dieser Ansatz einen Lösungsvorschlag für die berichtete Off-target Toxizität von multivalenten hochaffinen proteinischen Bindern, wie TAS266, darstellen, da hierin eine höhere Anzahl an Bindern mit niedriger Affinität verwendet wird. Dies könnte zu einer geringeren Retention auf gesunden Zellen mit geringerer DR5 Expressionsdichte und somit einer höheren Verträglichkeit in vivo führen, die zusätzlich durch Einführung von Bispezifität erhöht werden könnte.

Der dritte Teil dieser Arbeit sollte einen detaillierten Überblick über die enzymatische Generierung von ADCs mittels Transglutaminase geben. Trotz zahlreicher Publikation von Reviews zur Generierung von ADCs fehlt bis heute ein Überblick, der die Anwendung von Transglutaminase für diese Therapeutika detailliert beschreibt. Aus diesem Grund gibt der verfasste Review einen Überblick über die an dieser Thematik forschenden Arbeitsgruppen und Firmen, als auch eine gründliche Übersicht der eingesetzten Methoden, der adressierten Konjugationsstellen oder -motive, Linkern und verwendeten Zytostatika. Dieser Review beschreibt und diskutiert zukunftsweisende Syntheserouten und techniken, die jüngsten Fortschritte sowie verbleibende Einschränkungen der mTG-unterstützten Generierung von ADCs. Zusätzlich wurde eine weitere Studie durchgeführt, die die Verwendung von Dextran als multivalentes Grundgerüst untermauert. Hierfür wurde eine vorläufige Studie zur Validierung von Dextran als Träger für multiple chelatierende Komplexbildner, die Ionen für die Radiobildgebung oder -therapie komplexieren können, durchgeführt. Hierfür wurde Dextran mit einer gewünschten Anzahl an 1,4,7,10-Tetraazacyclododecan-1,4,7,10-tetraessigsäure (DOTA) beladen. In einer photometrischen Analyse von komplexierten Cu2+-Ionen konnte gezeigt werden, dass die resultierenden DOTA-Dextrankonjugate 3.2 respektive 5.3 Metallionen pro Polysaccharidgerüst tragen. Zurzeit werden die hergestellten DOTA-Dextrankonjugate in Biodistributionsstudien in Mäusen untersucht. Diese Konzeptstudien werden zeigen, ob diese neuen Konstrukte für in vivo Anwendungen geeignet sind und ob eine Konjugation an derzeit verwendete spezielle Bindungsproteine (Affibodies), welche in radioaktiv markierter Form Probleme mit der Hydrophobizität und daher mit Solubilität, Aggregation und Präzipitation haben, vorteilhaft ist. Dieser Ansatz bestärkt die Vermutung, dass Dextran ein vielversprechendes Gerüst für die mehrwertige Befestigung und Anpassung verschiedener Liganden darstellt.

Deutsch
URN: urn:nbn:de:tuda-tuprints-115020
Sachgruppe der Dewey Dezimalklassifikatin (DDC): 500 Naturwissenschaften und Mathematik > 540 Chemie
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
Fachbereich(e)/-gebiet(e): 07 Fachbereich Chemie
07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie
07 Fachbereich Chemie > Clemens-Schöpf-Institut > Fachgebiet Biochemie > Allgemeine Biochemie
Hinterlegungsdatum: 05 Apr 2020 19:55
Letzte Änderung: 05 Apr 2020 19:55
PPN:
Referenten: Kolmar, Prof. Dr. Harald ; Schmitz, Prof. Dr. Katja
Datum der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 10 Februar 2020
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