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

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 the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. 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 coassociating 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 Escherichia 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 coassembled 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 killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

Synthesis and biological evaluation of 2.4 nm thiolate-protected gold nanoparticles conjugated to Cetuximab for targeting glioblastoma cancer cells via the EGFR.

Therapeutic monoclonal antibodies benefit to patients and the conjugation to gold nanoparticles (AuNPs) might bring additional activities to these macromolecules. However, the behavior of the conjugate will largely depend on the bulkiness of the AuNP and small sizes are moreover preferable for diffusion. Water-soluble thiolate-protected AuNPs having diameters of 2-3 nm can be synthesized with narrow polydispersity and can selectively react with incoming organic thiols via a SN2-like mechanism. We therefore synthesized a mixed thionitrobenzoic acid- , thioaminobenzoic acid-monolayered AuNP of 2.4 nm in diameter and developed a site-selective conjugation strategy to link the AuNP to Cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody used in clinic. The water-soluble 80 kDa AuNP was fully characterized and then reacted to the hinge area of Cetuximab, which was selectively reduced using mild concentration of TCEP. The conjugation proceeded smoothly and could be analyzed by polyacrylamide gel electrophoresis, indicating the formation of a 1:1 AuNP-IgG conjugate as the main product. When added to EGFR expressing glioblastoma cells, the AuNP-Cetuximab conjugate selectively bound to the cell surface receptor, inhibited EGFR autophosphorylation and entered into endosomes like Cetuximab. Altogether, we describe a simple and robust protocol for a site-directed conjugation of a thiolate-protected AuNP to Cetuximab, which could be easily monitored, thereby allowing to assess the quality of the product formation. The conjugated 2.4 nm AuNP did not majorly affect the biological behavior of Cetuximab, but provided it with the electronic properties of the AuNP. This offers the ability to detect the tagged antibody and opens application for targeted cancer radiotherapy.

Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells.

Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome.

Generation of an intrabody-based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells.

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single-chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti-PCNA scFvs were screened in vitro after being tagged with dimeric glutathione-S-transferase. Anti-PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error-prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C-terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide-conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody-based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy.

Small p53 derived peptide suitable for robust nanobodies dimerization.

Bivalent VHHs have been shown to display better functional affinity compared with their monovalent counterparts. Bivalency can be achieved either by inserting a hinge region between both VHHs units or by using modules that lead to dimerization. In this report, a small self-associating peptide originating from the tetramerization domain of p53 was developed as a tool for devicing nanobody dimerization. This E3 peptide was evaluated for the dimerization of an anti-eGFP nanobody (nano-eGFP-E3) whose activity was compared to a bivalent anti-eGFP constructed in tandem using GS rich linker. The benefit of bivalency in terms of avidity and specificity was assessed in different in vitro and in cellulo assays. In ELISA and SPR, the dimeric and tandem formats were nearly equivalent in terms of gain of avidity compared to the monovalent counterpart. However, in cellulo, the nano-eGFP-E3 construct showed its superiority over the tandem format in terms of specificity with a highest and better ratio signal-to-noise. All together, the E3 peptide provides a universal suitable tool for the construction of dimeric biomolecules, in particular antibody fragments with improved functional affinity.

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

Advances in microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders originating from the immune response arsenal to gold nanoparticles (AuNPs) as contrasting agents is the method of choice to obtain labeling tools. However, conjugation of the minimal sized 15 kDa nanobody (Nb) to AuNPs remains challenging in comparison to the conjugation of 150 kDa IgG to AuNPs. Herein, effective Nb-AuNP assemblies are built using the selective and almost irreversible non-covalent associations between two peptide sequences deriving from a p53 heterotetramer domain variant. The 15 kDa GFP-binding Nb is fused to one dimerizing motif to obtain a recombinant Nb dimer with improved avidity for GFP while the other complementing dimerizing motif is equipped with thiols and grafted to a 2.4 nm substituted thiobenzoate-coordinated AuNP via thiolate exchange. After pegylation, the modified AuNPs are able to non-covalently anchor Nb dimers and the subsequent complexes demonstrate the ability to form immunogold label GFP-protein fusions within various subcellular locations. These tools open an avenue for precise localization of targets at high resolution by electron microscopy.

A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress.

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress.

Phosphorylated histone H2AX (γ-H2AX), a central player in the DNA damage response (DDR), serves as a biomarker of DNA double-strand break repair. Although DNA damage is generally visualized by the formation of γ-H2AX foci in injured nuclei, it is unclear whether the widespread uniform nuclear γ-H2AX (called pan-nuclear) pattern occurring upon intense replication stress (RS) is linked to DDR. Using a novel monoclonal antibody that binds exclusively to the phosphorylated C-terminus of H2AX, we demonstrate that H2AX phosphorylation is systematically pan-nuclear in cancer cells stressed with RS-inducing drugs just before they die. The pan-nuclear γ-H2AX pattern is abolished by inhibition of the DNA-PK kinase. Cell death induction of cancer cells treated with increasing combinations of replication and kinase (ATR and Chk1) inhibitory drugs was proportional to the appearance of pan-nuclear γ-H2AX pattern. Delivery of labeled anti-γ-H2AX Fabs in stressed cells demonstrated at a single cell level that pan-nuclear γ-H2AX formation precedes irreversible cell death. Moreover, we show that H2AX is not required for RS-induced cell death in HeLa cells. Thus, the nuclear-wide formation of γ-H2AX is an incident of RS-induced cell death and, thus, the pan nuclear H2AX pattern should be regarded as an indicator of lethal RS-inducing drug efficacy.

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm.

BACKGROUND: Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus. RESULTS: We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the alphabeta tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed in vivo in human cells since they were essentially localized in the nucleus. CONCLUSION: This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.

Single-chain Fv fragment antibodies selected from an intrabody library as effective mono- or bivalent reagents for in vitro protein detection.

In spite of their many potential applications, recombinant antibody molecules selected by phage display are rarely available commercially, one reason being the absence of robust bacterial expression systems that yield sufficient quantities of reagents for routine applications. We previously described the construction and validation of an intrabody library that allows the selection of single-chain Fv (scFv) fragments solubly expressed in the cytoplasm. Here, we show that it is possible to obtain monomeric scFvs binding specifically to human papillomavirus type 16 E6 and cellular gankyrin oncoproteins in quantities higher than 0.5 g/L of shake-flask culture in E. coli cytoplasm after auto-induction. In addition, stable bivalent scFvs of increased avidity were produced by tagging the scFvs with the dimeric glutathione-S-transferase enzyme (GST). These minibody-like molecules were further engineered by fusion with green fluorescent protein (GFPuv), leading to high yield of functional bivalent fluorescent antibody fragments. Our results demonstrate that scFvs selected from an intrabody library can be engineered into cost-effective bivalent reagents suitable for many biomedical and industrial applications.