Guilhem Chaubet

Antibody drug-conjugates (ADC) are a well-established class of therapeutics primarily used in oncology to selectively deliver highly cytotoxic agents into cancer cells. While ADC should theoretically spare healthy tissues and diminish side effects in patients, off-target toxicity is still observed, all the more serious as the drugs are extremely potent. In the quest towards safer payloads, we used the conventional chemotherapeutic drug vincristine to develop antibody-vincristine conjugates. Vincristine was N-alkylated with a cleavable linker and the resulting linker-payload conjugated to free cysteines of antibodies. We show that trastuzumab-vincristine conjugates display subnanomolar potency in vitro on HER2-positive cells, two orders of magnitude lower than free vincristine and comparable with marketed ADC. In vivo, trastuzumabvincristine conjugates led to remarkable efficacy when compared to two standards of care, with complete tumor regression just nine days after single administration. This highlights the untapped potential of the chemotherapeutic arsenal towards the development of novel ADC.

<div><p>Protocol to generate, purify, and analyze antibody-oligonucleotide conjugates from offthe-shelf antibodies Antibody-oligonucleotide conjugates (AOCs) are a fast-expanding modality for targeted delivery of therapeutic oligonucleotides to tissues. Here, we present a protocol to generate, purify, and analyze AOCs from off-the-shelf antibodies. We describe steps to conjugate single/doublestranded oligonucleotides bearing amine handles to linkers and, then, to antibodies using wellestablished chemistry. In addition, we provide details regarding the purification techniques and analytical methods suitable for AOC. This protocol can be applied for several purposes where AOC is a modality of interest.</p></div>

Studying the complex and intricate retinoids metabolic pathways by chemical biology approaches requires design and synthesis of biologically functional molecular probes. Only few of such molecular retinoid probes could be found in literature, most of them bearing a molecular structure quite different from natural retinoids. To provide close‐to‐native retinoid probes, we have developed a versatile late‐stage method for the insertion of azide function at the C4 position of several retinoids. This one‐step process opens straightforward access to different retinoid and carotenoid probes from commercially available precursors. We have further demonstrated that the different molecular probes retain ability of the original compound to activate genes’ transcription, despite azide insertion, highlighting biological activities that were further validated in zebrafish in vivo model. The present work paves the way to future studies on vitamin A's metabolism.

Disulfide rebridging methods are emerging recently as new ways to specifically modify antibody-based entities and produce future conjugates. Briefly, the solvent-accessible disulfide bonds of antibodies or antigen-binding fragments (Fab) thereof are reduced under controlled conditions and further covalently attached with a rebridging agent allowing the incorporation of one payload per disulfide bond. There are many examples of successful rebridging cases providing homogeneous conjugates due to the use of symmetrical reagents, such as dibromomaleimides. However, partial rebridging due to the use of unsymmetrical ones, containing functional groups with different reactivity, usually leads to the development of heterogeneous species that cannot be identified by a simple sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) due to its lack of sensitivity, resolution and low mass accuracy. Mass spectrometry coupled to liquid chromatography (LC-MS) approaches have already been demonstrated as highly promising alternatives for the characterization of newly developed antibody-drug-conjugate (ADC) and monoclonal antibody (mAb)-based formats. We report here the in-depth characterization of covalently rebridged antibodies and Fab fragments in-development, using size-exclusion chromatography hyphenated to mass spectrometry in denaturing conditions (denaturing SEC-MS, dSEC-MS). DSEC-MS was used to monitor closely the rebridging reaction of a conjugated trastuzumab, in addition to conjugated Fab fragments, which allowed an unambiguous identification of the covalently rebridged products along with the unbound species. This all-in-one approach allowed a straightforward analysis of the studied samples with precise mass measurement; critical quality attributes (CQAs) assessment along with rebridging efficiency determination.

The chemical bioconjugation of proteins has seen tremendous applications in the past decades, with the booming of antibody-drug conjugates and their use in oncology. While genetic engineering has permitted to produce bespoke proteins featuring key (un−)natural amino acid residues poised for site-selective modifications, the conjugation of native proteins is riddled with selectivity issues. Chemoselective strategies are plentiful and enable the precise modification of virtually any residue with a reactive side-chain; site-selective methods are less common and usually most effective on small and medium-sized proteins. In this context, we studied the application of the Ugi multicomponent reaction for the site-selective conjugation of amine and carboxylate groups on proteins, and antibodies in particular. Through an in-depth mechanistic methodology work supported by peptide mapping studies, we managed to develop a set of conditions allowing the highly selective modification of antibodies bearing N-terminal glutamate and aspartate residues. We demonstrated that this strategy did not alter their affinity toward their target antigen and produced an antibody-drug conjugate with subnanomolar potency. Excitingly, we showed that the high site selectivity of our strategy was maintained on other protein formats, especially on anticalins, for which directed mutagenesis helped to highlight the key importance of a single lysine residue.

Double-stranded RNAs (dsRNA)-based strategies appeared as promising therapies to induce an inflammation in the tumor microenvironment. However, currently described systems generally lack active targeting of tissues, and their clinical translation is thus limited to intratumoral injection. Herein, we developed an antibody-siRNA-5′triphosphate conjugate with multiple modes of action, combining cell surface EphA2-specific internalization, leading to a simultaneous gene silencing and activation of the receptor retinoic acid-inducible gene I (RIG-I). Recognition of cytosolic siRNA-5′triphosphate by RIG-I triggers the expression of interferons and pro-inflammatory cytokines, inducing an inflammation of the tumor environment and activating neighboring immune cells. In addition, these RIG-I-specific effects synergized with siRNA-mediated PLK1 silencing to promote cancer cell death by apoptosis. Altogether, such immune-stimulating antibody-RNA conjugate opens a novel modality to overcome some limitations encountered by dsRNA molecules currently in clinical trials.

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, μg, 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.

Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet’s oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant’s hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG4 linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.

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.

Peptide and protein bioconjugation sees ever‐growing applications in the pharmaceutical sector. Novel strategies and reagents that can address the chemo‐ and regioselectivity issues inherent to these biomolecules, while delivering stable and functionalizable conjugates, are therefore needed. Herein, we introduce the crosslinking ethynylbenziodazolone (EBZ) reagent JW‐AM‐005 for the conjugation of peptides and proteins through the selective linkage of cysteine residues. This easily accessed compound gives access to peptide conjugates or stapled peptides under mild and tuneable conditions. Applied to the antibody fragment of antigen binding (Fab) species, JW‐AM‐005 delivered rebridged proteins in a one‐pot three‐reaction process with high regioselectivity, outperforming the standard reagents commonly used for this transformation.

Cleavable linkers have become the subject of intense study in the field of chemical biology, particularly because of their applications in the construction of antibody-drug conjugates (ADC), where they facilitate lysosomal cleavage and liberation of drugs from their carrier protein. Due to lysosomes' acidic nature, acid-labile motifs have attracted much attention, leading to the development of hydrazone and carbonate linkers among several other entities. Continuing our efforts in designing new moieties, we present here a family of cyclic acetals that exhibit excellent plasma stability and acid lability, notably in lysosomes. Incorporated in ADC, they led to potent constructs with picomolar potency in vitro and similar in vivo efficacy as the commercially available ADC Kadcyla in mouse xenograft models.

The bioconjugation of proteins—that is, the creation of a covalent link between a protein and any other molecule—has been studied for decades, partly because of the numerous applications of protein conjugates, but also due to the technical challenge it represents. Indeed, proteins possess inner physico-chemical properties—they are sensitive and polynucleophilic macromolecules—that make them complex substrates in conjugation reactions. This complexity arises from the mild conditions imposed by their sensitivity but also from selectivity issues, viz the precise control of the conjugation site on the protein. After decades of research, strategies and reagents have been developed to address two aspects of this selectivity: chemoselectivity—harnessing the reacting chemical functionality—and site-selectivity—controlling the reacting amino acid residue—most notably thanks to the participation of synthetic chemistry in this effort. This review offers an overview of these chemical bioconjugation strategies, insisting on those employing native proteins as substrates, and shows that the field is active and exciting, especially for synthetic chemists seeking new challenges.

Antibody-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 antibodies’ 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.

Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN alcohol) is the most prominent strained-alkyne scaffold in chemical biology. Described herein is the synthesis of an oxidized analogue-BCN acidwhose facile functionalization via amide bond formation yields more stable derivatives than the classically encountered carbamates.

We report the unexpected acceleration of strain-promoted azide–alkyne cycloaddition in human plasma compared to classical solvent systems. Besides fast kinetics, human plasma also allows for discrimination between two azides in competition reaction.

A new alkaloid, manniindole 1, together with four known compounds: aristolactam AII 2, aristolactam BII 3, piperolactam D 4 and polycarpol 5 were isolated from the crude extract EtOHH2O (8:2) of the roots of Anonidium mannii by chromatographic separation. The structure elucidation was performed on the basis of a spectroscopic analysis (IR, HRESI MS, 1D and 2D NMR) as well as a comparison of their spectral data with those reported in the literature. For the first time, the crude extract and those isolated compounds were evaluated for their antischistosomal activity against Schistosoma mansoni and for cytotoxicity activity against Huh7 and A549 cells. Furthermore, they were also tested in vitro on the recent characterized Schistosoma mansoni NADþ catabolizing enzyme (SmNACE) for their impact on this enzyme which is localized on the outer surface of the adult parasite. Compound 2 displayed quite good worm killing capability, while 4 showed significant inhibition of SmNACE

Context: Anonidium mannii (Annonaceae) has been traditionally used in Africa to treat stomach aches, schistosomiasis, and many other illnesses. However, few phytochemical study and no investigation on schistosomiasis have been conducted on this species. This neglected tropical disease, caused by a worm, comes second after malaria as the most devastating parasitical infection. Aim: The goal of this study was to evaluate the anti-Schistosoma mansoni activity of fractions and constituents from A. mannii's stem bark and also to search efficient inhibitors of a recently discovered ectoenzyme of S. mansoni (S. mansoni nicotinamide adenine dinucleotide + catabolizing enzyme [SmNACE]). Materials and Methods: The powdered stem bark of A. mannii was extracted with ethanol/distilled water (80:20). The extract was then subjected to a partial bioguided separation by chromatography means. The structures of compounds were elucidated using modern spectroscopic techniques. Furthermore, isolated and semisynthetic compounds were evaluated for their antischistosomal and cytotoxic activities. Results: Chemical investigation led to the isolation and identification of eight compounds, in the majority, obtained for the first time from this genus. In addition, acetylation reactions were carried out to afford a new semisynthetic derivative. Preliminary biological screening of the extracts and compounds showed very good activities from antiparasitic and enzymatic tests and also very good percentage of cell viability evaluation. Conclusion: Like praziquantel drug, gallic acid exhibited full anthelmintic activity at concentration of 100 µM. On the other hand, piperolactam D showed important inhibition on SmNACE (IC 50 10 µM). Thus, standardization of bioactive fraction can help in improving traditional medicine. The optimization of those two compounds will enhance their selectivity/ effectiveness and could be used as seed for the development of new remedies against schistosomiasis. Further, the study will be focus on other pathogens species of Schistosoma genus.

Efficient methods to introduce bioorthogonal groups, such as terminal alkynes, into biomolecules are important tools for chemical biology. State-of-the-art approaches are based on the introduction of a linker between the targeted amino acid and the alkyne, and still present limitations of either reactivity, selectivity or adduct stability. Herein, we present an ethynylation method of cysteine residues based on the use of ethynylbenziodoxolone (EBX) reagents. In contrast to other approaches, the acetylene group is directly introduced onto the thiol group of cysteine and can be used in one-pot in a copper-catalyzed alkyne-azide cycloaddition (CuAAC) for further functionalization. Labeling proceeded with reaction rates comparable or higher than the most often used iodoacetamide on peptides or maleimide on the antibody trastuzumab. Under optimized conditions, high cysteine selectivity was observed. The reagents were also used in living cells for cysteine proteomic profiling and displayed an improved coverage of the cysteinome compared to previously reported iodoacetamide or hypervalent iodine-reagent based probes. Fine-tuning of the EBX reagents allowed optimization of their reactivity and physical properties for the desired application.

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the large majority of existing methods that solely target a single residue. We opted for the Ugi four-center three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads, and limits the potential conjugation sites to only six on the model antibody trastuzumab. Posttranslational modifications of proteins is Nature's way of generating a rich and diverse proteome from a more limited genetic coding capability. First occurrences of intentional, man-made-artificial-proteins modifications using a defined chemical-thus excluding the food-related Maillard reaction for example-could be dated back to the use of formaldehyde in the tanning industry or for the production of toxoids, 1,2 which evolved later on to immunization studies using chemically-modified bovine serum albumin in the 1900s and eventually led to Landsteiner's synthetic haptenes studies. 3,4 The field of protein modification has since largely benefited from the understanding of proteins' and amino acids' structures coupled to the parallel appearance of more efficient and precise analytical tools. This finally resulted in the development of bioconjugation reagents with excellent chemoselectivity towards various amino acids' side chains groups (i.e. residue-selectivity) that translated into major applications, notably in the pharmaceutical field with the generation of protein-fluorophore adducts for trafficking studies, or the polyethyleneglycol chains functionalization (PEGylation) of proteins to give less-immunogenic and more plasma-stable conjugates. 5,6 However, site selectivity quickly emerged as the main limitation of chemoselective strategies, due to the presence of multiple copies of each type of amino acid residue at the surface of proteins. Statistic conjugation of surface-accessible lysine residues with amine-selective reagents typically results in highly heterogeneous mixtures, containing up to millions of different adducts when large proteins such as antibodies are utilised. 7,8 Each of these adducts possessing distinct physicochemical properties, such chemoselective conjugation necessarily leads to mixtures with different in-vivo pharmacokinetic properties along with virtually no reproducibility in batch-to-batch production. 9,10 Regioselective (i.e., site-specific) methods were thus developed and are currently dominated by the use of recombinant proteins, incorporating exogenous amino acid residues-natural or unnatural-or peptide sequences that can be specifically targeted by a tailored reagent or strategy. 11-13 In parallel, site-selective chemical strategies for the conjugation of native and natural proteins have also flourished over the past few years, giving rise to methods targeting various types of amino acids-e.g. lysine, cysteine, tryptophan, tyrosine-that proved to be effective on proteins of all sorts of sizes, including antibodies. 14-28 With the aim of pursuing the efforts in this field, we could not help but notice that the vast majority of previously reported strategies for the site-selective conjugation of native proteins were focused on the modification of a unique residue. We hypothesized that targeting two different amino acid side chains simultaneously would lower the enormous subset of possibilities given by single-residue bioconjugation techniques, thus increasing our chances of developing a site-selective method by minimising the number

Here, we introduce 4-azidophenyl glyoxal (APG) as an efficient plug-and-play reagent for the selective functionalisation of arginine residues in native antibodies. The selective reaction between APG and arginines’ guanidine groups allowed a facile introduction of azide groups on the monoclonal antibody trastuzumab (plug stage). These pre-functionalised antibody–azide conjugates were then derivatised during the “play stage” via a biorthogonal cycloaddition reaction with different strained alkynes. This afforded antibody-fluorophore and antibody–oligonucleotide conjugates, all showing preserved antigen selectivity and high stability in human plasma. Due to a lower content of arginines compared to lysines in native antibodies, this approach is thus attractive for the preparation of more homogeneous conjugates. This method proved to be orthogonal to classical lysine-based conjugation and allowed straightforward generation of dual-payload antibody.

A new ring contraction of fused bis-lactams into spirolactams is presented here. In the presence of a triflate catalyst in various solvents under microwave irradiation, this rearrangement allows a clean conversion of some fused bicycles into spirocycles with good yields. The interest of this work thus lies in the use of activated 2,5-diketopiperazines as starting materials and demonstrates the wide range of applications of ring contraction reactions

Bis-Boc-activated 2,5-diketopiperazines on reaction with potassium hydroxide or sodium methoxide in dry tetrahydrofuran led to Boc-protected hydantoins through an unprecedented ring contraction. This rearrangement was applied to several monosubstituted 2,5-diketopiperazines with good yields and regioselectivity.

Access to diastereoisomeric forms of original spirolactam frameworks and investigation of their folded potentials are depicted here. Taking advantage of a stereoselective ring-contraction reaction, the Transannular Rearrangement of Activated Lactams (TRAL), followed by two unprecedented tandem reactions, we describe here an efficient access to elegant spirocyclic scaffolds. After dimerization, NMR analyses, circular dichroism, SEM and molecular modelling indicated the existence of an attractive edifice able to fold and behave as a PPII helix, a common yet neglected peptidic secondary structure.

A tandem aza-FriedeleCrafts reaction/Hantzsch cyclization is described to access various polysubstituted 2-amino-1,3-thiazoles from electron-rich (hetero)-aromatic rings, aldehydes, thiourea and a-chloroketones.

Ces travaux de thèse s'inscrivent dans un projet à long terme concernant le développement de nouvelles réactions de contraction de cycle originales afin d'accéder à des édifices moléculaires organisés à activités biologiques potentielles. Généralement découvertes de manière fortuite, les réactions de contraction de cycle sont des réarrangements offrant l'avantage de modifier rapidement le squelette des molécules et permettant donc un accès facile à des analogues structurels, une propriété intéressante et utile aussi bien en chimie de synthèse qu'en chimie médicinale. Dans cette optique, trois réactions de contraction de cycle différentes ainsi que leurs applications seront rapportées dans ce manuscrit. La première décrit la réactivité particulière des bis-Boc 2,5-dicétopipérazines (DKPs) en milieu basique et leur conversion en hydantoïnes, deux squelettes hétérocycliques d'intérêt pharmacologique. Ce nouveau réarrangement a été appliqué à différentes DKPs avec des rendements satisfaisants et de bons excès énantio- ou diastéréoiso-mériques. L'intérêt des bis-Boc DKPs en tant que plateforme de départ dans la construction de structures complexes a ensuite été démontré lors de l'obtention de spirolactames grâce à l'utilisation de la réaction de réarrangement transannulaire de lactames activés (TRAL) et la mise au point d'une stratégie de cyclisation rapide et efficace. Après dimérisation de ces bicycles, les études par dichroïsme circulaire, RMN et modélisation moléculaire ont mis en évidence un comportement similaire à celui d'hélices de polyproline II (PPII), des structures peptidiques secondaires largement impliquées dans les interactions protéine-protéine et dans des processus pathogènes. Afin de valider le potentiel de mimes de nos dimères, une fonctionnalisation de ces substrats s'est avérée nécessaire, qui a été en partie réalisée grâce à la découverte d'une nouvelle réaction de contraction de cycle. Effectuée à chaud dans plusieurs solvants et en présence d'un catalyseur de type triflate, ce réarrangement permet la conversion de quelques bicycles accolés en spirocycles avec de bons rendements. L'intérêt de ces travaux réside ainsi sur l'utilisation de 2,5-dicétopipérazines activées comme substrat de départ et démontre la gamme d'applications multiples des réactions de contraction de cycle.