Pirzer, Thomas ; Becher, Kira-Sophie ; Rieker, Marcel ; Meckel, Tobias ; Mootz, Henning D. ; Kolmar, Harald (2018)
Generation of Potent Anti-HER1/2 Immunotoxins by Protein Ligation Using Split Inteins.
In: ACS chemical biology, 13 (8)
Artikel, Bibliographie

Kurzbeschreibung (Abstract)

Cell targeting protein toxins have gained increasing interest for cancer therapy aimed at increasing the therapeutic window and reducing systemic toxicity. Because 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. Here, we describe the production of full-length antibody immunotoxins using self-splicing split inteins. 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 trastuzumab, a human epidermal growth factor receptor 2 (HER2) receptor targeting antibody and to a nanobody-Fc fusion targeting the HER1 receptor, respectively. Both antibodies were produced in Expi293F cells. The longer C-terminal counterpart of the intein was genetically fused to the protein toxins gelonin or Pseudomonas Exotoxin A, respectively, and expressed in E. coli via fusion to maltose binding protein. Using optimized in vitro splicing conditions, we were able to generate a set of specific and potent immunotoxins with IC values in the mid- to subpicomolar range.

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