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Intein mediated high throughput screening for bispecific antibodies

Hofmann, Tim Lothar (2020):
Intein mediated high throughput screening for bispecific antibodies. (Publisher's Version)
Darmstadt, Technische Universität,
DOI: 10.25534/tuprints-00013280,
[Ph.D. Thesis]

Abstract

The plethora of bispecific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions, spanning from enhanced selectivity by simultaneous avid binding to distinct effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Pharmaceutical antibody discovery has been evolving and continuously growing over the past decades, moving towards the field of complex biologics and mostly bispecific antibodies. Despite their high potential value, discovery of bispecific antibodies as the identification of the best possible combination of two parental monospecific antibodies, however, remains challenging. Discovery of two sets of monospecific antibodies followed by cloning, production and functional investigation of combinations is tedious and often resulting in undesired extended development times and increased expenses. But although automated high throughput screening approaches have become increasingly relevant and mature for pharmaceutical small molecule and classical antibody discovery, screening of bispecific antibodies is, however, up to now very limited by laborious preparation of the tremendous number of potential bispecific combinations. Therefore, a novel high throughput screening method for bispecific antibodies was developed in this study, allowing a full coverage of the large combinatorial screening space and bypass the afore mentioned limitations. This achievement is realized by the ability of the split intein Npu DnaE, to splice proteins in trans. Antibody fragments are fused within the hinge region to a respective split intein part, capable to reconstitute two antibody fragments back to a full-length antibody format in vitro, without extensive cloning and manufacturing work. Throughout the study, all reconstituted antibodies remained similarly biologically active in several biochemical and functional cell assays when compared to genetically fused references. The reconstitution method is furthermore amenable for automated high throughput screening providing the possibility to screen for bispecific combinations by combinatorial mixing of antibody fragments. High throughput amenability was investigated for 96 well and 384 well plates confirming both high reconstitution efficiency and reproducibility. Fab fragments were combined with different Fc fragments as exemplary application for fast switch effector function screenings of monoclonal in addition to bispecific antibodies. The method described could enable bispecific antibody high throughput binding and functional cellular screenings to greatly shorten development times and enhance the probability of identifying the optimal combination, ultimately leading to the generation of better biotherapeutics.

Item Type: Ph.D. Thesis
Erschienen: 2020
Creators: Hofmann, Tim Lothar
Status: Publisher's Version
Title: Intein mediated high throughput screening for bispecific antibodies
Language: English
Abstract:

The plethora of bispecific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions, spanning from enhanced selectivity by simultaneous avid binding to distinct effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Pharmaceutical antibody discovery has been evolving and continuously growing over the past decades, moving towards the field of complex biologics and mostly bispecific antibodies. Despite their high potential value, discovery of bispecific antibodies as the identification of the best possible combination of two parental monospecific antibodies, however, remains challenging. Discovery of two sets of monospecific antibodies followed by cloning, production and functional investigation of combinations is tedious and often resulting in undesired extended development times and increased expenses. But although automated high throughput screening approaches have become increasingly relevant and mature for pharmaceutical small molecule and classical antibody discovery, screening of bispecific antibodies is, however, up to now very limited by laborious preparation of the tremendous number of potential bispecific combinations. Therefore, a novel high throughput screening method for bispecific antibodies was developed in this study, allowing a full coverage of the large combinatorial screening space and bypass the afore mentioned limitations. This achievement is realized by the ability of the split intein Npu DnaE, to splice proteins in trans. Antibody fragments are fused within the hinge region to a respective split intein part, capable to reconstitute two antibody fragments back to a full-length antibody format in vitro, without extensive cloning and manufacturing work. Throughout the study, all reconstituted antibodies remained similarly biologically active in several biochemical and functional cell assays when compared to genetically fused references. The reconstitution method is furthermore amenable for automated high throughput screening providing the possibility to screen for bispecific combinations by combinatorial mixing of antibody fragments. High throughput amenability was investigated for 96 well and 384 well plates confirming both high reconstitution efficiency and reproducibility. Fab fragments were combined with different Fc fragments as exemplary application for fast switch effector function screenings of monoclonal in addition to bispecific antibodies. The method described could enable bispecific antibody high throughput binding and functional cellular screenings to greatly shorten development times and enhance the probability of identifying the optimal combination, ultimately leading to the generation of better biotherapeutics.

Place of Publication: Darmstadt
Collation: IV, 121 Seiten
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Fachgebiet Biochemie
Date Deposited: 10 Dec 2020 10:16
DOI: 10.25534/tuprints-00013280
Official URL: https://tuprints.ulb.tu-darmstadt.de/13280
URN: urn:nbn:de:tuda-tuprints-132809
Referees: Kolmar, Prof. Dr. Harald ; Hust, Prof. Dr. Michael
Refereed / Verteidigung / mdl. Prüfung: 20 July 2020
Alternative Abstract:
Alternative abstract Language

Bispezifische Antikörper können verschiedene molekulare Architekturen umfassen, um Wirkungsmechanismen zu erzeugen, die durch monospezifische Antikörper nicht adressiert werden können. Diese Wirkungsmechanismen beinhalten unter anderem die Rekrutierung von Effektorzellen oder die erhöhte Selektivität durch zielgerichtete gleichzeitige Bindung von zwei Antigenen. Die Antikörperforschung in der Pharmaindustrie ist in den letzten Jahren kontinuierlich gewachsen, besonders bispezifische Antikörper mit ihren besonderen Eigenschaften stehen im Fokus. Allerdings ist deren Entwicklung sehr herausfordernd, da die optimale Kombination aus zwei ursprünglich monospezifischen Antikörpern gefunden werden muss. Daher kommt es zu unerwünscht verlängerten Entwicklungszeiten und erhöhtem Kostenaufwand. Für Identifikation und Charakterisierung von Kleinmolekülen und klassischen monoklonalen Antikörpern ist mittlerweile das Hochdurchsatz-Durchmustern eine ausgereifte Disziplin. Die Bereitstellung der sehr hohen Anzahl möglicher bispezifischer Kombinationen für ein solches Vorgehen stellt bis jetzt jedoch einen sehr limitierenden Faktor dar. Es kann nur ein kleiner Teil des zu durchmusternden Raumes abgedeckt werden, da die verschiedenen Kombinationen erst aufwendig einzeln neu kloniert, exprimiert und gereinigt werden müssen. In der vorliegenden Studie wird daher ein neuartiger Hochdurchsatz-Durchmusterungsansatz für bispezifische Antikörper vorgestellt, der diese Limitierungen umgeht und den Durchmusterungsraum um ein Vielfaches erweitert. Er basiert auf der Fähigkeit des Split Inteins Npu DnaE, Proteine in-trans zu spleißen. Antikörperfragmente werden dabei in der Hinge-Region an einen Teil des jeweiligen Split Inteins fusioniert, um die zwei Fragmente so in vitro kombinatorisch ligieren zu können. Diese Methode erlaubt die Rekonstitution einer großen Anzahl verschiedenster bispezifischer Antikörper in kürzester Zeit unter voll automatisierten Bedingungen ohne aufwendige Einzelklonierungen und Herstellungsarbeiten. Verschiedene in dieser Arbeit durch Rekonstitution hergestellte Antikörper zeigen im Vergleich zu genetisch fusioniert hergestellten Referenzen durchweg vergleichbare Bindeverhalten und funktionelle Eigenschaften. Die erarbeitete Rekonstitutionsmethode ist außerdem voll implementierungsfähig für automatisierte HochdurchsatzDurchmusterung. Eine potenzielle Hochdurchsatz Zugänglichkeit für 96-Well und 384-Well Platten wurde untersucht und bestätigt und diente als konzeptioneller Beweis für die Funktionsfähigkeit der Methode. FabFragmente wurden mit verschiedenen Fc-Fragmenten kombiniert, als beispielhafte Screening Anwendung für einen schnellen Wechsel der Effektor-Funktion von monoklonalen als auch bispezifischen Antikörpern. Die beschriebene Methode könnte es ermöglichen, bispezifische Antikörper im Hochdurchsatz auf Bindung und zelluläre Funktionalität zu screenen, um die Entwicklungszeiten stark zu verkürzen und die Wahrscheinlichkeit eine optimale bispezifische Kombination zu finden, erhöhen. Diese Methode dient letztlich zur Herstellung besserer Biotherapeutika.

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