Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides

Abstract

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell targeting protein combined to a polypeptide toxin.Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect activity and/or stability of the conjugate proteins and additional purification steps are often required to isolate the final conjugate from unwanted by-products. As for fusion proteins, they often suffer from low solubility and yield.In this report, we describe a straightforward conjugation process to generate an immunotoxin using co-associating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa Exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in E. coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the co-assembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2 overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to kill HER2-overexpressing breast cancer cells with IC 50 values in the picomolar range. Altogether, we demonstrate that this non-covalent conjugation method using two co-assembling peptides can be easily implemented for modular engineering of immunotoxins targeting different types of cancers.

Small p53 derived peptide suitable for robust nanobodies dimerization

“…Because V H Hs display unique properties, such as high solubility and a reversible unfolding reaction under stringent conditions (Muyldermans, 2013), E3 small peptide is of particular interest for dimerization of V H Hs. Indeed, we have previously reported that E3 peptide is robust at low pH, form in complex media and resists thermal denaturation above 90°C (Stoessel et al, 2020;Vigneron et al, 2019), making it a powerful tool for bivalent V H Hs engineering.…”

“…We have recently taken advantage of a modified tetramerization domain of p53 to construct bifunctional and bispecific heterotetramer biomolecules (Stoessel et al, 2020;Vigneron et al, 2019). This tetramerization domain (p53tet) is a small peptide of 31-aminoacids, which can be fused to various moieties.…”

“…obvious detection differences between the monovalent and the bivalent formats. These interactions were specific, as the nano-HER2 used as a negative control (Stoessel et al, 2020), did not bind to H2B-eGFP and thus localized in both cytoplasmic and nuclear compartments. For the MTS-eGFP localized in the mitochondria compartment, the transduced V H Hs clearly co-localized with the eGFP signal except for the negative control nano-HER2, as expected (Figure 5A right panel).…”

Gold labelling of a green fluorescent protein (GFP)-tag inside cells using recombinant nanobodies conjugated to 2.4 nm thiolate-coated gold nanoparticles

“…17 These E-and K-enriched sequences, named E3 and K3 respectively, were recently demonstrated to conveniently favour the selective non-covalent union of 2 distinctly tagged functional proteins into highly stable heterotetramers even in complex biological fluids crowded with all sorts of proteins and diverse compounds. 18,33 First, the Nb was genetically engineered at its C-terminal end with the aspartate-enriched variant of the p53 heterotetramer domain (E3) along with a polyhistidine sequence (Figure 2). 18 The recombinant Nb-E3 fusion protein was then produced in E. coli and purified by immobilized metal affinity chromatography followed by size exclusion chromatography (SEC) with high yield and purity (Figure 2B).…”

“…30 To design Nb-AuNP conjugates with usefulness for gold immunolabelling application of cellular specimen, we explored methods for strongly linking thionitrobenzoates-coated AuNPs (AuG) 31,32 to bioengineered GFP binding Nbs. 8,20 We discovered that the non-covalent conjugation approach based on the complementary associating dimers originating from a p53 heterotetramer variant, 17,18,33 led to Nb:AuNP assemblies with excellent immunogold labelling abilities (Figure 1).…”