TU Darmstadt / ULB / TUbiblio

Generation of anti-HER1/2 immunotoxins by protein ligation using split inteins

Pirzer, Thomas (2018):
Generation of anti-HER1/2 immunotoxins by protein ligation using split inteins.
Darmstadt, Technische Universität, [Online-Edition: https://tuprints.ulb.tu-darmstadt.de/7754],
[Ph.D. Thesis]

Abstract

Cell targeting protein toxins have gained increasing interest for cancer therapy, aimed at increasing the therapeutic window and reducing systemic toxicity. Since recombinant expression of immunotoxins consisting of a receptor-binding and a cell-killing moiety is hampered by their high toxicity in a eukaryotic production host, most applications rely on recombinant production of fusion proteins consisting of an antibody fragment and a protein toxin in bacterial hosts such as Escherichia coli (E. coli). These fusions often lack beneficial properties of whole antibodies like extended serum half-life or efficient endocytic uptake via receptor clustering. This work aimed to generate novel immunotoxins composed of full-length antibodies and different toxins by protein trans-splicing using split inteins. Initially, different toxins were optimized for expression by testing a variety of expression hosts, induction parameters and fusion partners. The plant toxin gelonin and variants of the bacterial Pseudomonas Exotoxin A were used and expressed in E. coli. Fusions to thioredoxin and maltose binding protein resulted in reproducible and acceptable yields. The HER2 binding antibody trastuzumab was used as a model for therapeutic antibodies with known properties. Additionally, a new antibody was designed, composed of two VHH domains that were attached in tandem on an IgG1 Fc scaffold, resulting in a heavy-chain antibody with specificity towards EGFR. Both antibodies were produced in mammalian cells at good yields. A split intein was used to connect both antibodies and toxins to form biologically active immunotoxins. To this end, the short (11 amino acids) N-terminal intein part of the artificially designed split intein M86, a derivative of the Ssp DnaB intein, was recombinantly fused to the heavy chain of the antibodies, while the longer (143 amino acids) C-terminal intein part was fused to the toxins. By mixing both reaction partners under reducing conditions, the intein assembled into its active form and splicing occurred. Reaction conditions for protein splicing were optimized in in vitro reactions. Parameters included concentration of reducing agents, time and aggregation state of the toxin. Both antibodies could be linked to gelonin and exotoxin A with splicing efficacies of 50 – 70 %. Generated immunotoxins were purified by protein A chromatography and immobilized metal affinity chromatography. The resulting constructs were characterized by a toxin/antibody ratio of about 1.3 and were analyzed in more detail. Specific cell binding was analyzed and confirmed for all immunotoxins. The activity of gelonin was confirmed by an in vitro translation assay with cell lysates. Confocal microscopy was used to follow Abstract 3 cellular uptake and confirmed endosomal uptake as well as cleavage of a protease-labile linker and subsequent translocation of the toxin out of the endosomes. All immunotoxins exhibited IC50 values in the mid- to subpicomolar range in cytotoxicity assays with different cell lines, numbering them among the most toxic immunotoxins reported to date.

Item Type: Ph.D. Thesis
Erschienen: 2018
Creators: Pirzer, Thomas
Title: Generation of anti-HER1/2 immunotoxins by protein ligation using split inteins
Language: English
Abstract:

Cell targeting protein toxins have gained increasing interest for cancer therapy, aimed at increasing the therapeutic window and reducing systemic toxicity. Since recombinant expression of immunotoxins consisting of a receptor-binding and a cell-killing moiety is hampered by their high toxicity in a eukaryotic production host, most applications rely on recombinant production of fusion proteins consisting of an antibody fragment and a protein toxin in bacterial hosts such as Escherichia coli (E. coli). These fusions often lack beneficial properties of whole antibodies like extended serum half-life or efficient endocytic uptake via receptor clustering. This work aimed to generate novel immunotoxins composed of full-length antibodies and different toxins by protein trans-splicing using split inteins. Initially, different toxins were optimized for expression by testing a variety of expression hosts, induction parameters and fusion partners. The plant toxin gelonin and variants of the bacterial Pseudomonas Exotoxin A were used and expressed in E. coli. Fusions to thioredoxin and maltose binding protein resulted in reproducible and acceptable yields. The HER2 binding antibody trastuzumab was used as a model for therapeutic antibodies with known properties. Additionally, a new antibody was designed, composed of two VHH domains that were attached in tandem on an IgG1 Fc scaffold, resulting in a heavy-chain antibody with specificity towards EGFR. Both antibodies were produced in mammalian cells at good yields. A split intein was used to connect both antibodies and toxins to form biologically active immunotoxins. To this end, the short (11 amino acids) N-terminal intein part of the artificially designed split intein M86, a derivative of the Ssp DnaB intein, was recombinantly fused to the heavy chain of the antibodies, while the longer (143 amino acids) C-terminal intein part was fused to the toxins. By mixing both reaction partners under reducing conditions, the intein assembled into its active form and splicing occurred. Reaction conditions for protein splicing were optimized in in vitro reactions. Parameters included concentration of reducing agents, time and aggregation state of the toxin. Both antibodies could be linked to gelonin and exotoxin A with splicing efficacies of 50 – 70 %. Generated immunotoxins were purified by protein A chromatography and immobilized metal affinity chromatography. The resulting constructs were characterized by a toxin/antibody ratio of about 1.3 and were analyzed in more detail. Specific cell binding was analyzed and confirmed for all immunotoxins. The activity of gelonin was confirmed by an in vitro translation assay with cell lysates. Confocal microscopy was used to follow Abstract 3 cellular uptake and confirmed endosomal uptake as well as cleavage of a protease-labile linker and subsequent translocation of the toxin out of the endosomes. All immunotoxins exhibited IC50 values in the mid- to subpicomolar range in cytotoxicity assays with different cell lines, numbering them among the most toxic immunotoxins reported to date.

Place of Publication: Darmstadt
Divisions: 07 Department of Chemistry
07 Department of Chemistry > Fachgebiet Biochemie
07 Department of Chemistry > Fachgebiet Biochemie > Allgemeine Biochemie
Date Deposited: 16 Sep 2018 19:55
Official URL: https://tuprints.ulb.tu-darmstadt.de/7754
URN: urn:nbn:de:tuda-tuprints-77542
Referees: Kolmar, Prof. Dr. Harald and Mootz, Prof. Dr. Henning D.
Refereed / Verteidigung / mdl. Prüfung: 2 August 2018
Alternative Abstract:
Alternative abstract Language
Zellspezifische Proteintoxine haben durch die Möglichkeiten, das therapeutische Fenster zu vergrößern und die systemische Toxizität zu senken, zunehmendes Interesse in der Krebstherapie geweckt. Aufgrund limitierender Toxizität in eukaryotischen Expressionssystemen, beruhten die meisten Anwendungen bisher auf der Fusion von Antikörperfragmenten mit Toxinen, die in Bakterien, wie Escherichia coli (E. coli) hergestellt werden können. Diesen Fusionen fehlen jedoch häufig nützliche Eigenschaften von Volllängenantikörpern, wie eine verlängerte Serumhalbwertszeit oder effiziente endosomale Aufnahme durch Rezeptorgruppierung. Ziel dieser Arbeit war die Herstellung neuartiger Immuntoxine, bestehend aus Volllängenantikörpern und verschiedenen Proteintoxinen, durch Proteinspleißen in trans mit geteilten Inteinen. Zunächst wurde die Toxinexpression durch Optimierung verschiedener Parameter, wie Expressionsstamm, Induktionskonditionen und Fusionspartnern, verbessert. Das pflanzliche Gelonin und das bakterielle Pseudomonas Exotoxin A wurden dafür in E. coli produziert. Fusionen mit Thioredoxin und Maltosebindeprotein erzielten reproduzierbare und akzeptable Ausbeuten. Der HER2 bindende Antikörper Trastuzumab wurde als Modell für therapeutisch relevante und gut beschriebene Antikörper verwendet. Zusätzlich wurde ein, aus zwei EGFR bindenden VHH Domänen bestehender, Antikörper entwickelt, welcher nur aus einer schweren Kette besteht. Beide Antikörper wurden in Säugetierzellen in guten Ausbeuten produziert. Ein geteiltes Intein wurde verwendet, um Antikörper und Toxine zu verbinden und daraus biologisch aktive Immuntoxine zu generieren. Der kurze (11 Aminosäuren) N-terminale Teil des aus Ssp DnaB evolvierten M86 Inteins wurde rekombinant an die Antikörper fusioniert, während der längere (143 Aminosäuren) Teil an die Toxine fusioniert wurde. Durch das Mischen der beiden Reaktionspartner konnte sich das Intein in seine aktive Form falten und das Spleißen ausführen. Die Reaktionsbedingungen wurden in diversen in vitro Reaktionen getestet und optimiert. Zu ihnen gehörten die Konzentration des Reduktionsmittels, die Reaktionszeit und der Aggregationsstatus der Toxine. Beide Antikörper konnten mit Spleißeffizienzen von 50 – 70 % erfolgreich mit Gelonin und Exotoxin A verbunden werden. Diese Immuntoxine wurden durch Protein A und immobilisierte Metallaffinitätschromatographie gereinigt. Die Endkonstrukte waren durch ein Toxin/Antikörper Verhältnis von 1,3 gekennzeichnet und wurden daraufhin detaillierter analysiert. Die spezifische Zellbindung konnte bei allen Immuntoxinen bestätigt werden. Außerdem wurde die enzymatische Aktivität von Gelonin in einem in vitro Translationsversuch bestätigt. Konfokalmikroskopie wurde angewandt, um die endosomale Aufnahme, die dort stattfindende Spaltung eines proteaselabilen Linkers und die anschließende Translokation des Toxins ins Zytoplasma zu bestätigen. Alle Immuntoxine Abstract 2 zeichneten sich in Zelltoxizitätsexperimenten auf verschiedenen Zelllinien durch IC50 Werte im mittleren pikomolaren bis hohen femtomolaren Bereich aus, wodurch sie zu den potentesten bisher publizierten Immuntoxinen gehören.German
Export:
Suche nach Titel in: TUfind oder in Google
Send an inquiry Send an inquiry

Options (only for editors)

View Item View Item