Développement de vecteurs innovants pour le ciblage d'antigènes cancéreux

Actuellement, les anticorps sont largement utilisés en thérapie pour le ciblage de cellules tumorales car ils présentent une haute affinité et spécificité pour leur cible. Cependant, ils présentent des limites pour une utilisation en imagerie, principalement liées à leur grande taille qui entraîne une biodistribution et une élimination lentes de l organisme. Ces inconvénients amènent au développement de vecteurs alternatifs aux anticorps. Ces dernières années, l équipe d accueil a utilisé une stratégie originale pour développer la nouvelle classe de protéines affines artificielles, les Affitins. Ce sont des variants de Sac7d, issue de l archée Sulfolobus acidocaldarius qui a pour rôle physiologique de fixer l ADN double brin. Elle présente de nombreuses propriétés attractives pour une alternative aux anticorps : 20 fois plus petite qu un anticorps conventionnel, soluble, thermiquement très stable et résiste aux pH extrêmes. Différentes cibles ont déjà été atteintes (protéines bactériennes, humaines et animales, bactéries vivantes) montrant qu il est possible d isoler des variants de Sac7d stables, solubles, avec un taux de production élevé chez Escherichia coli et capables de se lier de manière spécifique à leur cible avec des affinités de l ordre du nanomolaire. Des premiers résultats in vivo montrent une biodistribution et une clairance rapide adaptée à une utilisation en imagerie. Le but de ma thèse était dans un premier temps de sélectionner des Affitins contre HER-1 et HER-2, récepteurs surexprimés à la surface de nombreuses cellules cancéreuses, marqueurs de mauvais pronostic car ils engendrent notamment une stimulation de la croissance du cancer et une inhibition de l apoptose. Dans un deuxième temps, mon travail a consisté à mettre au point un système qui permettra de cribler les Affitins sélectionnées directement sur des cellules exprimant HER1 et HER2.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Application of Affitins for Affinity Purification of Proteins

International audienc

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

Whole-bacterium ribosome display selection for isolation of highly specific anti-Staphyloccocus aureus Affitins for detection- and capture- based biomedical applications

International audienceDetection and capture methods using antibodies have been developed to ensure identification of pathogens in biological samples. Though antibodies have many attractive properties, they also have limitations and there are needs to expand the panel of available affinity proteins with different properties. Affitins, that we developed from the Sul7d proteins, are a solid class of affinity proteins, which can be used as substitutes to antibodies or to complement them. We report the generation and characterization of antibacterial Affitins with high specificity for Staphylococcus aureus. For the first time, ribosome display selections were carried out using whole-living-cell and naïve combinatorial libraries, which avoid production of protein targets and immunization of animals. We showed that Affitin C5 exclusively recognizes S. aureus among dozens of strains, including clinical ones. C5 binds staphylococcal Protein A (SpA) with a K D of 108 ± 2 nM and has a high thermostability (T m  = 77.0°C). Anti-S. aureus C5 binds SpA or bacteria in various detection and capture applications, including ELISA, western blot analysis, bead-fishing, and fluorescence imaging. Thus, novel anti-bacteria Affitins which are cost-effective, stable, and small can be rapidly and fully designed in vitro with high affinity and specificity for a surface-exposed marker. This class of reagents can be useful in diagnostic and biomedical applications

Multivalent Affidendrons with High Affinity and Specificity toward Staphylococcus aureus as Versatile Tools for Modulating Multicellular Behaviors

International audienceMultivalency is a widely occurring natural phenomenon often exploited in nanotechnology to enhance biorecognition. We report the preparation and characterization of versatile, multivalent Affitin-dendrimer conjugates (Affidendrons) showcased by a set targeting Staphylococcus aureus (S. aureus), an opportunistic pathogen causing numerous hospital-and community-acquired infections. Affitins are small affinity proteins characterized by higher stability and lower cost-effective production than antibodies. The strategy presented provides a platform for the rational design of multivalent nanodevices that, retaining the ability of Affitins to recognize their target with high specificity, achieve a largely enhanced affinity. Affidendrons with precisely designed size and valency have been exploited to modulate complex multicellular behaviors of S. aureus, such as agglutination and biofilm formation. Agglutination assays showed that Affidendrons rapidly cross-link S. aureus strains with high bacterial cell selectivity. Moreover, remarkably low concentrations of Affidendrons were able to effectively prevent biofilm formation. Overall, Affidendrons represent a promising platform for the rapid and selective pathogen identification, infection imaging, and theranostic applications

Tolerance of the archaeal Sac7d scaffold protein to alternative library designs: characterization of anti-immunoglobulin G Affitins

International audienceEngineered protein scaffolds have received considerable attention as alternatives to antibodies in both basic and applied research, as they can offer superior biophysical properties often associated with a simpler molecular organization. Sac7d has been demonstrated as an effective scaffold for molecular recognition. Here, we used the initial L1 'flat surface' library constructed by randomization of 14 residues, to identify ligands specific for human immunoglobulin G. To challenge the plasticity of the Sac7d protein scaffold, we designed the alternative L2 'flat surface & loops' library whereof only 10 residues are randomized. Representative binders (Affitins) of the two libraries exhibited affinities in the low nanomolar range and were able to recognize different epitopes within human immunoglobulin G. These Affitins were stable up to pH 12 while largely conserving other favorable properties of Sac7d protein, such as high expression yields in Escherichia coli, solubility, thermal stability up to 80.7°C, and acidic stability (pH 0). In agreement with our library designs, mutagenesis study revealed two distinct binding areas, one including loops. Together, our results indicate that the Sac7d scaffold tolerates alternative library designs, which further expands the diversity of Affitins and may provide a general way to create tailored affinity tools for demanding applications

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)

Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins

International audienceAffinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals