Pirzer, Thomas (2018)
Generation of anti-HER1/2 immunotoxins by protein ligation using split inteins.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (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.

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