A novel generation of Affitins for targeting cancer cells with drug-loaded nanocapsules

A progressive strategy against cancer is the targeting of tumour-associated antigens by specific ligands coupled to nanoparticles, carrying therapeutic or imaging agents. Antibodies are the most widely used targeting molecules, but they possess limitations as high production costs, complex structure and limited stability. Affitins are highly stable engineered affinity proteins, derived originally from Sac7d, an archaeal polypeptide from the 7 kDa DNA-binding family (also known as Sul7d family). These binders show comparable affinity and specificity to those of antibodies, while being thermally and chemically more stable, cheaper to produce, easier to engineer and present a simpler structure and 20-fold smaller size. Lipid nanocapsules (LNCs), prepared by solvent free process, possess great stability and high efficiency for lipophilic drugs encapsulation and protect the drug from rapid degradation. Targeting drug-LNC to cancer cells can further decrease drug concentration in normal tissues and lower the toxicity. The aim of the project is to combine the advantages of Affitins as targeting agents and LNCs as carriers in order to create vehicles for delivering payloads to cancer cells. The first goal of this work was to identify and characterize a shorter member, but still very stable, of the Sul7d family in order to further improve the affinity scaffold, and then to use it for the generation of Affitins, recognizing the tumour-associated Epithelial Cell Adhesion Molecule. The last goal was to attach the new binders as affinity moieties to LNCs and to assess the tumour targeting of these complexes.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 201

Use of Affitins for the development of functionalized nanoparticles to deliver payloads to targeted colorectal tumor cells

Une approche prometteuse contre le cancer est le ciblage d’antigènes associés aux tumeurs par des ligands spécifiques couplés à des nanoparticules transportant des agents thérapeutiques ou d’imagerie. Les anticorps sont les molécules de ciblage les plus utilisées, mais présentent des limitations en termes de coûts de production élevés, de complexité structurale et de stabilité limitée. Les Affitins sont des protéines d'affinité hautement stables, dérivées à l'origine de Sac7d, un polypeptide d’archée de la famille de liaison à l'ADN de 7 kDa (Sul7d). Les Affitins montrent une affinité et une spécificité comparables à celles des anticorps, tout en étant thermiquement et chimiquement plus stable, moins coûteuses à produire, plus faciles à remodeler avec une taille 20 fois plus petite. Les nanocapsules lipidiques (LNC) possèdent une grande stabilité et une efficacité élevée pour l'encapsulation des médicaments lipophiles et les protègent de la dégradation rapide. Le ciblage de cellules cancéreuses par des LNC peut réduire de plus la concentration de médicament dans les tissus normaux et réduire la toxicité. Le but du projet est de combiner les avantages des Affitins et des LNC pour amener les médicaments anticancéreux vers les cellules cancéreuses. Le premier objectif a été d'identifier et de caractériser un membre plus court, mais toujours très stable, de la famille Sul7d pour encore améliorer la base moléculaire des Affitins, puis de générer des Affitins qui reconnaissant le biomarqueur EpCAM associé aux cellules tumorales. Le deuxième objectif a été d'attacher les nouvelles Affitins comme ligands d'affinité aux LNC et d'évaluer le ciblage de la tumeur par ces complexes.A progressive strategy against cancer is the targeting of tumour-associated antigens by specific ligands coupled to nanoparticles, carrying therapeutic or imaging agents. Antibodies are the most widely used targeting molecules, but they possess limitations as high production costs, complex structure and limited stability. Affitins are highly stable engineered affinity proteins, derived originally from Sac7d, an archaeal polypeptide from the 7 kDa DNA-binding family (also known as Sul7d family). These binders show comparable affinity and specificity to those of antibodies, while being thermally and chemically more stable, cheaper to produce, easier to engineer and present a simpler structure and 20-fold smaller size. Lipid nanocapsules (LNCs), prepared by solvent free process, possess great stability and high efficiency for lipophilic drugs encapsulation and protect the drug from rapid degradation. Targeting drug-LNC to cancer cells can further decrease drug concentration in normal tissues and lower the toxicity. The aim of the project is to combine the advantages of Affitins as targeting agents and LNCs as carriers in order to create vehicles for delivering payloads to cancer cells. The first goal of this work was to identify and characterize a shorter member, but still very stable, of the Sul7d family in order to further improve the affinity scaffold, and then to use it for the generation of Affitins, recognizing the tumour-associated Epithelial Cell Adhesion Molecule. The last goal was to attach the new binders as affinity moieties to LNCs and to assess the tumour targeting of these complexes

The archaeal "7kDa DNA-binding" proteins: extended characterization of an old gifted family

International audienceThe " 7 kDa DNA-binding " family, also known as the Sul7d family, is composed of chromatin proteins from the Sulfolobales archaeal order. Among them, Sac7d and Sso7d have been the focus of several studies with some characterization of their properties. Here, we studied eleven other proteins alongside Sac7d and Sso7d under the same conditions. The dissociation constants of the purified proteins for binding to double-stranded DNA (dsDNA) were determined in phosphate-buffered saline at 25 °C and were in the range from 11 μM to 22 μM with a preference for G/C rich sequences. In accordance with the extremophilic origin of their hosts, the proteins were found highly stable from pH 0 to pH 12 and at temperatures from 85.5 °C to 100 °C. Thus, these results validate eight putative " 7 kDa DNA-binding " family proteins and show that they behave similarly regarding both their function and their stability among various genera and species. As Sac7d and Sso7d have found numerous uses as molecular biology reagents and artificial affinity proteins, this study also sheds light on even more attractive proteins that will facilitate engineering of novel highly robust reagents. In living organisms, the long genomic DNA has to be packed in order to fit into cells, while the genetic information must stay accessible for replication and transcription events. To this aim, organisms have developed different compaction systems, such as the wrapping of DNA around histones to form the chromatin in Eukarya, and the supercoiling of DNA with the help of non-histone proteins to form the nucleoid in Bacteria. Archaea often live in extreme environments and have the additional challenge to protect their genomic DNA from extreme conditions, such as high temperatures. Many Archaea contain homologs of eukaryotic histones, but Desulfurococcales, Thermoplasmatales and Sulfolobales use a different kind of packaging proteins 1,2. Hyperthermophile and acidophile archaea of the Sulfolobales order from the Crenarchaeota kingdom express small basic DNA-binding proteins, which represent about 5% of the total soluble cellular proteins, sufficient to coat the entire genome of a Sulfolobus cell 3. These proteins constitute the family called " 7 kDa DNA-binding " or Sul7d 4. They were first isolated from Sulfolobus acido-caldarius which produces five of them, named Sac7a, b, c, d, and e. Sac7d and Sac7e are encoded by distinct genes, while Sac7a and b are truncated versions of Sac7d 5–7. Highly similar homologs have been found in all Sulfolobus species, such as Sso7d from Sulfolobus solfataricus 8 , and Ssh7a and Ssh7b from Sulfolobus shibatae-two proteins encoded by two distinct genes 3,9. Sac7d and Sso7d are the two most studied proteins of this family. They have been characterized for their structure, function, chemical stability and biophysical properties 7. Sac7d and Sso7d are hyperthermostable (T m = 90.4 °C and 100.2 °C, respectively) 10,11 and are resistant from pH 0 up to at least pH 12 12,13. Although Sac7d and Sso7d sequences show only few differences, Sso7d is more stable than Sac7d. Their three-dimensional structures show that they both fold as an SH3-like domain capped by a C-terminal α-helix 14,15 and that they sharply kink the double DNA helix upon binding into the minor groove 16,17. It has been shown that Sac7d and Sso7d are general dsDNA binders with K D values varying in a salt dependent manner from 20 nM (low salt) to 3.8 μ M (high salt) for Sac7d, and from 116 nM to 12.8 μ M for Sso7d, and with a preference for G/C rich sequences 18,19. Sac7d has the property to increase the thermal stability of DNA duplexes by as much as 43.5 °C 6,15. Furthermore, Ssh7a and Ssh7b have been partially characterized and an affinity for dsDNA of about 100 n

Targeted radionuclide therapy with astatine-211: Oxidative dehalogenation of astatobenzoate conjugates

International audience211 At is a most promising radionuclide for targeted alpha therapy. However, its limited availability and poorly known basic chemistry hamper its use. Based on the analogy with iodine, labelling is performed via astatobenzoate conjugates, but in vivo deastatination occurs, particularly when the conjugates are internalized in cells. Actually, the chemical or biological mechanism responsible for deastatination is unknown. In this work, we show that the C−At "organometalloid" bond can be cleaved by oxidative dehalogenation induced by oxidants such as permanganates, peroxides or hydroxyl radicals. Quantum mechanical calculations demonstrate that astatobenzoates are more sensitive to oxidation than iodobenzoates, and the oxidative deastatination rate is estimated to be about 6 × 10 6 faster at 37 °C than the oxidative deiodination one. Therefore, we attribute the "internal" deastatination mechanism to oxidative dehalogenation in biological compartments, in particular lysosomes

A novel, smaller scaffold for Affitins: Showcase with binders specific for EpCAM.

Affitins are highly stable engineered affinity proteins, originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from Sulfolobus genera. Their efficiency as reagents for intracellular targeting, enzyme inhibition, affinity purification, immunolocalization, and various other applications has been demonstrated. Recently, we have characterized the 7 kDa DNA-binding family, and Aho7c originating from Acidianus hospitalis was shown to be its smallest member with thermostability comparable to those of Sac7d and Sso7d. Here, after four rounds of selection by ribosome display against the human recombinant Epithelial Cell Adhesion Molecule (hrEpCAM), we obtained novel Aho7c-based Affitins. The binders were expressed in soluble form in Escherichia coli, displayed high stability (up to 74°C; pH 0-12) and were shown to be specific for the hrEpCAM extracellular domain with picomolar affinities (KD  = 110 pM). Thus, we propose Aho7c as a good candidate for the creation of Affitins with a 10% smaller size than the Sac7d-based ones (60 vs. 66 amino acids)