Citation: Mielecki, M.; Ziemniak, M.; Ozga, M.; Borowski, R.; Antosik, J.; Kaczy ´ nska, A.; Paj ˛ ak, B. Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin. Biomolecules 2022, 12, 1111. https://doi.org/10.3390/ biom12081111 Academic Editor: Robert J. Kreitman Received: 7 July 2022 Accepted: 9 August 2022 Published: 12 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Article Structure–Activity Relationship of the Dimeric and Oligomeric Forms of a Cytotoxic Biotherapeutic Based on Diphtheria Toxin Marcin Mielecki † , Marcin Ziemniak † , Magdalena Ozga, Radoslaw Borowski, Jaroslaw Antosik, Angelika Kaczy ´ nska and Beata Paj ˛ ak * WPD Pharmaceuticals, ˙ Zwirki and Wigury 101, 02-089 Warsaw, Poland * Correspondence: beata.pajak@wpdpharmaceuticals.com † These authors contributed equally to this work. Abstract: Protein aggregation is a well-recognized problem in industrial preparation, including biotherapeutics. These low-energy states constantly compete with a native-like conformation, which is more pronounced in the case of macromolecules of low stability in the solution. A better un- derstanding of the structure and function of such aggregates is generally required for the more rational development of therapeutic proteins, including single-chain fusion cytotoxins to target specific receptors on cancer cells. Here, we identified and purified such particles as side products of the renaturation process of the single-chain fusion cytotoxin, composed of two diphtheria toxin (DT) domains and interleukin 13 (IL-13), and applied various experimental techniques to comprehensively understand their molecular architecture and function. Importantly, we distinguished soluble purified dimeric and fractionated oligomeric particles from aggregates. The oligomers are polydisperse and multimodal, with a distribution favoring lower and even stoichiometries, suggesting they are composed of dimeric building units. Importantly, all these oligomeric particles and the monomer are cystine-dependent as their innate disulfide bonds have structural and functional roles. Their reduction triggers aggregation. Presumably the dimer and lower oligomers represent the metastable state, retaining the native disulfide bond. Although significantly reduced in contrast to the monomer, they preserve some fraction of bioactivity, manifested by their IL-13RA2 receptor affinity and selective cytotoxic potency towards the U-251 glioblastoma cell line. These molecular assemblies probably pre- serve structural integrity and native-like fold, at least to some extent. As our study demonstrated, the dimeric and oligomeric cytotoxin may be an exciting model protein, introducing a new understanding of its monomeric counterpart’s molecular characteristics. Keywords: biotherapeutics; cytotoxin; diphtheria toxin; IL-13; inclusion bodies; refolding; disulfide bond; protein oligomerization; MALS; SAXS; LC/MS 1. Introduction Immunotoxins (IT) are fusion biotherapeutics composed of two major parts: a receptor- binding moiety and the active toxic payload. The binding part is usually an antibody or a ligand directed towards a specific receptor expressed on the cell membrane, such as IL-13RA2. The active part of the IT is the toxic payload, such as drugs, radioisotopes, toxins, and enzymes [1]. Compared to most drugs, toxins act catalytically, do not develop drug resistance, and can be applied to both dividing and quiescent cells [2]. A major advantage of toxins, when compared to radionucleotides and many small molecule drugs, is a lack of unspecific toxicity to surrounding cells and easier handling. The latter is the main drawback of radiopharmaceuticals [3]. The most commonly used toxins are of plant origin, for example, gelonin and ricin [4], or from bacteria, such as diphtheria toxin (DT) and exotoxin A (PE), produced by Corynebacterium diphtheriae and Pseudomonas aeruginosa, respectively [5]. Both DT and PE have shown to be highly efficient in killing eukaryotic cells. These molecules can irreversibly modify the mammalian elongation factor Biomolecules 2022, 12, 1111. https://doi.org/10.3390/biom12081111 https://www.mdpi.com/journal/biomolecules