10 research outputs found
Novel targeted delivery systems through non-covalent assemblies of antibody-oligonucleotide conjugates
Les outils de la bioconjugaison permettent aujourdâhui de dĂ©velopper des composĂ©s chimĂ©riques Ă partir de quasiment nâimporte quelle combinaison de molĂ©cules biologiques et synthĂ©tiques. Un exemple de ce type de combinaison innovante se trouve dans les conjuguĂ©s anticorps-oligonuclĂ©otide, qui bĂ©nĂ©ficient Ă la fois de la grande capacitĂ© de reconnaissance des anticorps et des nombreuses propriĂ©tĂ©s des oligonuclĂ©otides, pouvant ĂȘtre utilisĂ©s soit pour leurs activitĂ©s biologiques soit pour leur rĂŽle structurant. Ces travaux sont consacrĂ©s Ă lâexploration des propriĂ©tĂ©s, des potentielles applications thĂ©rapeutiques et de la synthĂšse de cette nouvelle famille de conjuguĂ©s. Dans une premiĂšre partie nous dĂ©crivons comment la fonctionnalisation dâun anticorps par des oligonuclĂ©otides modifie sa capacitĂ© Ă reconnaĂźtre son antigĂšne de maniĂšre spĂ©cifique. Dans une seconde partie, nous utilisons lâaffinitĂ© des nanoparticules polycationiques envers les oligonuclĂ©otides afin de former des complexes conjuguĂ©s anticorps-oligonuclĂ©otide/nanoparticule, et Ă©valuons le potentiel de ces derniers en tant que systĂšmes de dĂ©livrance ciblĂ©e (dans lesquels la nanoparticule sert non pas de vecteur mais dâagent cytotoxique) que nous avons appelĂ©s Anticorps-Nanoparticules toxiques. Enfin, nous prĂ©sentons une nouvelle mĂ©thode pour synthĂ©tiser des conjuguĂ©s anticorps-oligonuclĂ©otide stoechiomĂ©triquement dĂ©finis Ă partir de faibles quantitĂ©s de produits de dĂ©part, et qui sâest Ă©galement montrĂ©e utile pour la synthĂšse de conjuguĂ©s anticorps-polymĂšres.The tools of bioconjugation chemistry have made it possible to engineer chimeric compounds with novel biological properties from virtually any combination of biological and synthetic molecules. One example of such innovative combination are antibody-oligonucleotide conjugates which benefit from both the high binding abilities of antibodies and the versatile properties of oligonucleotides, which can be used either for their biological activity or their role as structuring elements. This work is devoted to exploring the properties, potential therapeutic applications, and synthesis of this emerging family of conjugates. The first part describes how appending oligonucleotides to antibodies can modify their ability to specifically target their antigen. In a second part, we used the affinity of polycationic nanoparticles for oligonucleotides to form antibody-oligonucleotide conjugates/nanoparticle complexes, and evaluated their potential as a novel type of targeted delivery system (where the nanoparticle act not as a carrier but as a cytotoxic compound) which we named Antibody-Toxic Nanoparticles. Finally, we present a new method to synthesize stoichiometrically defined antibodyoligonucleotide conjugates from small amounts of starting materials, which also proved useful to synthesize antibody-polymer conjugates
Non-specific interactions of antibody-oligonucleotide conjugates with living cells
International audienceAntibody-Oligonucleotide Conjugates (AOCs) represent an emerging class of functionalized antibodies that have already been used in a wide variety of applications. While the impact of dye and drug conjugation on antibodiesâ ability to bind their target has been extensively studied, little is known about the effect caused by the conjugation of hydrophilic and charged payloads such as oligonucleotides on the functions of an antibody. Previous observations of non-specific interactions of nucleic acids with untargeted cells prompted us to further investigate their impact on AOC binding abilities and cell selectivity. We synthesized a series of single- and double-stranded AOCs, as well as a human serum albumin-oligonucleotide conjugate, and studied their interactions with both targeted and non-targeted living cells using a time-resolved analysis of ligand binding assay. Our results indicate that conjugation of single strand oligonucleotides to proteins induce consistent non-specific interactions with cell surfaces while double strand oligonucleotides have little or no effect, depending on the preparation method
Direct Cytosolic Delivery of Citraconylated Proteins
Current intracellular protein delivery strategies face the challenge of endosomal entrapment and consequent degradation of protein cargo. Methods to efficiently deliver proteins directly to the cytosol have the potential to overcome this hurdle. Here, we report the use of a straightforward approach of protein modification using citraconic anhydride to impart an overall negative charge on the proteins, enabling them to assemble with positively charged nano vectors. This strategy uses anhydride-modified proteins to electrostatically form polymerâprotein nanocomposites with a cationic guanidinium-functionalized polymer. These supramolecular self-assemblies demonstrated the efficient cytosolic delivery of modified proteins through a membrane fusion-like mechanism. This approach was validated on five cell lines and seven proteins as cargo. Retention of protein function was confirmed through efficient cell killing via the intracellular enzymatic activity of RNase A. This platform provides a versatile, straightforward, and single-step method of protein modification and efficient direct cytosolic protein delivery
Hexavalent thiofucosides to probe the role of the Aspergillus fumigatus lectin FleA in fungal pathogenicity
International audienceA. fumigatus is a pathogenic fungus infecting the respiratory system and responsible for a variety of life-threatening lung diseases. A fucose-binding lectin named FleA which has a controversial role in A. fumigatus pathogenesis was recently identified. New chemical probes with high affinity and enzymatic stability are needed to explore the role of FleA in the infection process. In this study, we developed potent FleA antagonists based on optimized and non-hydrolysable thiofucoside ligands. We first synthesized a set of monovalent sugars showing micromolar affinity for FleA by isothermal titration calorimetry. The most potent derivative was co-crystallized with FleA to gain insights into the binding mode in operation. Its chemical multimerization on a cyclodextrin scaffold led to an hexavalent compound with a significantly enhanced binding affinity (Kd = 223 ± 21 nM) thanks to a chelate binding mode. The compound could probe the role of bronchial epithelial cells in a FleA-mediated response to tissue invasio
Multivalent Fucosides with Nanomolar Affinity for the Aspergillus fumigatus Lectin FleA Prevent Spore Adhesion to Pneumocytes
FleA (or AFL), a fucose lectin, was recently identified in the opportunistic mold Aspergillus fumigatus, which causes fatal lung infections in immunocompromised patients. We designed di-, hexa- and octavalent fucosides with various spacer arm lengths to block the hexameric FleA through chelation. Microcalorimetry measurements showed that the ethylene glycol (EG) spacer arm length has a strong influence on the binding affinity of the divalent fucosides. The relationship between the EG length and chelate binding efficiency to FleA was explored according to polymer theory. Hexa- and octavalent compounds based on cyclodextrin and octameric silsesquioxane scaffolds were nanomolar FleA inhibitors, surpassing their monovalent fucose analogue by more than three orders of magnitude. Importantly, some of the fucosides were highly efficient in preventing fungal spore adhesion to bronchoepithelial cells, with half maximal inhibitory concentration values in the micromolar range. We propose that the synergistic antiadhesive effect observed can be ascribed to chelate binding to FleA and to the formation of conidium aggregates, as observed by optical microscopy. These fucosides are promising tools that can be used to better understand the role of FleA in conidia pathogenicity and host defenses against invasive aspergillosis
Targeted Anticancer Agent with Original Mode of Action Prepared by Supramolecular Assembly of Antibody Oligonucleotide Conjugates and Cationic Nanoparticles
Despite their clinical success, Antibody-Drug Conjugates (ADCs) are still limited to the delivery of a handful of cytotoxic small-molecule payloads. Adaptation of this successful format to the delivery of alternative types of cytotoxic payloads is of high interest in the search for novel anticancer treatments. Herein, we considered that the inherent toxicity of cationic nanoparticles (cNP), which limits their use as oligonucleotide delivery systems, could be turned into an opportunity to access a new family of toxic payloads. We complexed anti-HER2 antibody-oligonucleotide conjugates (AOC) with cytotoxic cationic polydiacetylenic micelles to obtain Antibody-Toxic-Nanoparticles Conjugates (ATNPs) and studied their physicochemical properties, as well as their bioactivity in both in vitro and in vivo HER2 models. After optimising their AOC/cNP ratio, the small (73 nm) HER2-targeting ATNPs were found to selectively kill antigen-positive SKBR-2 cells over antigen-negative MDA-MB-231 cells in serum-containing medium. Further in vivo anti-cancer activity was demonstrated in an SKBR-3 tumour xenograft model in BALB/c mice in which stable 60% tumour regression could be observed just after two injections of 45 pmol of ATNP. These results open interesting prospects in the use of such cationic nanoparticles as payloads for ADC-like strategies
On the use of DNA as a linker in antibody-drug conjugates: synthesis, stability and in vitro potency
International audienceHere we present the synthesis and evaluation of antibody-drug conjugates (ADCs), for which antibody and drug are non-covalently connected using complementary DNA linkers. These ADCs are composed of trastuzumab, an antibody targeting HER2 receptors overexpressed on breast cancer cells, and monomethyl auristatin E (MMAE) as a drug payload. In this new ADC format, trastuzumab conjugated to a 37-mer oligonucleotide (ON) was prepared and hybridized with its complementary ON modified at 5-end with MMAE (cON-MMAE) in order to obtain trastuzumab-DNA-MMAE. As an advantage, the cON-MMAE was completely soluble in water, which decreases overall hydrophobicity of toxic payload, an important characteristic of ADCs. The stability in the human plasma of these non-engineered ON-based linkers was investigated and showed a satisfactory half-life of 5.8 days for the trastuzumab-DNA format. Finally, we investigated the in vitro cytotoxicity profile of both the DNA-linked ADC and the ON-drug conjugates and compared them with classical covalently linked ADC. Interestingly, we found increased cytotoxicity for MMAE compared to cON-MMAE and an EC50 in the nanomolar range for trastuzumab-DNA-MMAE on HER2-positive cells. Although this proved to be less potent than classically linked ADC with picomolar range EC50, the difference in cytotoxicity between naked payload and conjugated payload was significant when an ON linker was used. We also observed an interesting increase in cytotoxicity of trastuzumab-DNA-MMAE on HER2-negative cells. This was attributed to enhanced non-specific interaction triggered by the DNA strand as it could be confirmed using ligand tracer assay
Reinvestigation of the Automated Synthesis of Stoichiometrically Conjugated Antibodies to Access High Molecular Weight Payloads and Multiplexed Conjugation via an In-Solution Trans-Tagging Process
Protein conjugates have found applications in a wide variety of fields, ranging from therapeutics to imaging and detection. However, robust control over the parameters of the conjugation process (such as sites and degree of conjugation) remains challenging. Previously, our group introduced Equimolar NAtive Chemical Tagging (ENACT), a method which allows for the monofunctionalization of proteins by combining an iterative low-conversion bioconjugation, an automated process, and a bioorthogonal trans-tagging reaction. However, while the automated ENACT was dimensioned to achieve monoconjugation at the mg scale, in early stage research, because of the rarity and cost of the starting materials, it is often necessary to prepare conjugates at the lower, ÎŒg, scale. Here, we introduce modified ENACT protocols, as well as a new ENACT conjugation reagent, which allow for the monofunctionalization of proteins on the micrograms scale, using minimal quantities of payload