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.
Antibodies have long been recognized as clinically relevant biomarkers of disease. The onset of a disease often stimulates antibody production in low quantities, making it crucial to develop sensitive, specific, and easy-to-use antibody assay platforms. Antibodies are also extensively used as probes in bioassays, and there is a need for simpler methods to evaluate specialized probes, such as antibody-oligonucleotide (AbO) conjugates. Previously, we demonstrated that thermofluorimetric analysis (TFA) of analyte-driven DNA assembly can be leveraged to detect protein biomarkers using AbO probes. A key advantage of this technique is its ability to circumvent autofluorescence arising from biological samples, which otherwise hampers homogeneous assays. The analysis of differential DNA melt curves (dF/dT) successfully distinguishes the signal from the background and interferences. Expanding the applicability of TFA further, herein we demonstrate a unique proximity based TFA assay for antibody quantification that is functional in 90% human plasma. We show that the conformational flexibility of the DNA-based proximity probes is critically important for optimal performance in these assays. To promote stable, proximity-induced hybridization of the short DNA strands, substitution of poly(ethylene glycol) (PEG) spacers in place of ssDNA segments led to improved conformational flexibility and sensor performance. Finally, by applying these flexible spacers to study AbO conjugates directly, we validate this modified TFA approach as a novel tool to elucidate the probe valency, clearly distinguishing between monovalent and multivalent AbOs and reducing the reagent amounts by 12-fold.
Immunoconjugates , Oligonucleotides , Humans , Antibodies , DNA/analysis , Proteins , DNA Probes
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.
OBJECTIVE: The aim: Assessment of the effectiveness of using minimally invasive and open methods of bile duct decompression for treatment of obstructive jaundice (OJ) by comparing complications in patients of di!erent age groups. PATIENTS AND METHODS: Materials and methods: We analyzed the results of surgical treatment of 250 patients with OJ. The patients were assigned to two groups: Group I (n = 100) consisting of young and middle-age patients, and Group II (n = 150) consisting of elderly, senile and long-living patients. The average age was 52 ± 6.0 years. RESULTS: Results: 62 (24.8%) Group I patients and 74 (29.6%) Group II patients were submitted to minimally invasive surgical interventions. 38 (15.2%) Group I patients and 76 (30.4%) Group II patients were submitted to open surgical interventions. Complications after minimally invasive surgery (n = 62) in Group I patients were observed in 2 (3.2%) cases, and in 4 (10.5%) cases after open surgeries (n = 38). Complications following minimally invasive interventions (n = 74) in Group II patients were registered in 5 (6.8%) cases, and in 9 (11.8%) cases following open operations (n = 76). 2 (2.6%) Group II patients died for transmural myocardial infarction. CONCLUSION: Conclusions: The use of minimally invasive surgical interventions for treatment of young and middle-aged OJ patients compared to patients of older age groups makes it possible to reduce the frequency of complications by 2.1 times, which is a statistically signi"cant (p <0.05). The frequency of complications after open surgical interventions of bile ducts in patients of di!erent age groups is not statistically signi"cant (p >0.05).
Jaundice, Obstructive , Aged , Middle Aged , Humans , Jaundice, Obstructive/etiology , Jaundice, Obstructive/surgery , Bile Ducts/surgery , Drainage/methods , Minimally Invasive Surgical Procedures
OBJECTIVE: The aim: To study structural disorders of the liver depending on the duration of subhepatic cholestasis in patients of different age groups. PATIENTS AND METHODS: Materials and methods: 50 obstructive jaundice patients were subdivided into two groups. Group I (n = 25) consisted of young (18-44-year-old) and middle-aged patients (45-59-year-old), while the Group II (n = 25) included elderly (60-74-year-old) and senile patients (75-90-year-old). RESULTS: Results: We performed morphological and morphometric studies of 50 liver biopsy specimens taken from patients of different age groups with different duration of obstructive jaundice: less than 7 days, 7-14 days, 14-21 days, 21-28 days, and over 28 days. CONCLUSION: Conclusions: In patients of the Groups I and II, pathological hepatic changes in the early stages of mechanical jaundice were manifested in the form of he-patocyte dystrophy and hepatitis development. In the Group I patients, manifestations of steatohepatitis, fibrosis and initial signs of liver cirrhosis were noted in the late stages of subhepatic cholestasis. In addition to the above-mentioned changes, Group II patients, in the late stages of mechanical jaundice, presented signs of severe fibrosis and well-shaped liver cirrhosis. Taking into account the above morphological changes in the liver with different duration of subhepatic cholestasis, we consider reasonable to decompress bile ducts in patients of older age groups at earlier stages of mechanical jaundice compared to young and middle-aged patients, thus preventing post-decompression liver dysfunction and the subsequent development of biliary cirrhosis.
Cholestasis , Jaundice, Obstructive , Adolescent , Adult , Aged , Aged, 80 and over , Humans , Infant, Newborn , Middle Aged , Young Adult , Bile Ducts , Cholestasis/complications , Jaundice, Obstructive/etiology , Liver Cirrhosis/etiology
Mediterranean spotted fever (MSF) has been diagnosed clinically in the Crimean Peninsula since the 1930s. We describe the recent illness of an elderly patient from Crimea who had developed a classic triad of MSF symptoms consisting of fever, maculopapular rash, and eschar. Clinical diagnosis of rickettsiosis was confirmed using real-time PCR and sequencing of 4 Rickettsia protein genes. The strain causing clinical illness was characterized as Rickettsia conorii subspecies conorii Malish 7. This report corroborates the utility of eschar swab material as a source of DNA for PCR-based diagnostics that enables timely patient treatment and management.
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 acid - whose facile functionalization via amide bond formation yields more stable derivatives than the classically encountered carbamates.
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 great majority of existing methods that solely target a single residue. We opted for the Ugi four-centre 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; furthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.
Immunoconjugates , Trastuzumab , Amino Acid Sequence , Amino Acids/chemistry , Antineoplastic Agents, Immunological/chemistry , Immunoconjugates/chemistry , Trastuzumab/chemistry
Here 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.
Antineoplastic Agents/chemistry , DNA , Immunoconjugates/chemistry , Oligopeptides/chemistry , Trastuzumab/chemistry , Cell Line, Tumor , Humans
Controlled protein functionalization holds great promise for a wide variety of applications. However, despite intensive research, the stoichiometry of the functionalization reaction remains difficult to control due to the inherent stochasticity of the conjugation process. Classical approaches that exploit peculiar structural features of specific protein substrates, or introduce reactive handles via mutagenesis, are by essence limited in scope or require substantial protein reengineering. We herein present equimolar native chemical tagging (ENACT), which precisely controls the stoichiometry of inherently random conjugation reactions by combining iterative low-conversion chemical modification, process automation, and bioorthogonal trans-tagging. We discuss the broad applicability of this conjugation process to a variety of protein substrates and payloads.
Antibody-oligonucleotide conjugates (AOCs) are a novel class of synthetic chimeric biomolecules that has been continually gaining traction in different fields of modern biotechnology. This is mainly due to the unique combination of the properties of their two constituents, exceptional targeting abilities and antibody biodistribution profiles, in addition to an extensive scope of oligonucleotide functional and structural roles. Combining these two classes of biomolecules in one chimeric construct has therefore become an important milestone in the development of numerous biotechnological applications, including imaging (DNA-PAINT), detection (PLA, PEA), and therapeutics (targeted siRNA/antisense delivery). Numerous synthetic approaches have been developed to access AOCs ranging from stochastic chemical bioconjugation to site-specific conjugation with reactive handles, introduced into antibody sequences through protein engineering. This Review gives a general overview of the current status of AOC applications with a specific emphasis on the synthetic methods used for their preparation. The reported synthetic techniques are discussed in terms of their practical aspects and limitations. The importance of the development of novel methods for the facile generation of AOCs possessing a defined constitution is highlighted as a priority in AOC research to ensure the advance of their new applications.
Antibodies/metabolism , Immunoconjugates/therapeutic use , Molecular Imaging/methods , Oligonucleotides/metabolism , Animals , Humans , Immunoconjugates/chemistry , Immunoconjugates/metabolism
The reduction-rebridging strategy is a powerful method for the preparation of stable and homogeneous antibody-drug conjugates (ADCs). In this communication, we describe the development of the arylene-dipropiolonitrile (ADPN) functional group for the rebridging of reduced disulphide bonds and its application in the preparation of potent and selective ADCs.
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.
Antibodies, Monoclonal/chemistry , Arginine/chemistry , Azides/chemistry , Phenylglyoxal/analogs & derivatives , Alkynes/chemistry , Cycloaddition Reaction , Immunoconjugates/chemistry , Lysine/chemistry , Phenylglyoxal/chemistry , Trastuzumab/chemistry
The biochemical characteristics of hetero-bifunctional cross-linkers used in bioconjugates are of essential importance to the desired features of the final adduct (i.e. antibody-drug conjugates). These include stability in biological media, chemical and biological reactivities, cleavability under defined conditions, and solubility. In our previous work, we introduced a new amino-to-thiol linker, maleimidomethyl dioxane (MD), as an alternative to classical maleimide conjugation, with increased hydrophilicity and serum stability due to succinimidyl ring-opening. In this work, we investigate the generality of linkers containing a dioxo-ring with regard to their ability to self-hydrolyze and their surprising stability at a low pH. We synthesized four FRET probes which allowed us to address the stability of the dioxo-ring and to study the maleimide ring-opening and the thiol-exchange processes by means of detecting and measuring the generation of fluorescence. It was found that the ring expansion (from a 5- to a 6-membered ring) improved the stability of the probes in aqueous media, and the increase of the chain length between the dioxo-ring and the succinimide ring (from methylene to ethylene) decreased the rate of succinimidyl ring-opening.
Maleimides/chemistry , Drug Stability , Humans , Hydrogen-Ion Concentration , Maleimides/blood , Models, Molecular , Protein Conformation , Pyrans/chemistry , Serum Albumin, Human/chemistry , Sulfhydryl Compounds/chemistry , Water/chemistry
We report a plug-and-play strategy for the preparation of functionally enhanced antibodies with a defined average degree of conjugation (DoC). The first stage (plug) allows the controllable and efficient installation of azide groups on lysine residues of a native antibody using 4-azidobenzoyl fluoride. The second step (play) allows for versatile antibody functionalization with a single payload or combination of payloads, such as a toxin, a fluorophore, or an oligonucleotide, via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC). It is notable that in comparison to a classical N-hydroxysuccinimide ester (NHS) strategy, benzoyl fluorides show faster and more efficient acylation of lysine residues in a PBS buffer. This translates into better control of the DoC and enables the efficient and fast functionalization of delicate biomolecules at low temperature.
Antibodies, Monoclonal/chemistry , Benzyl Compounds/chemistry , Fluorides/chemistry , Immunoconjugates/chemistry , Lysine/chemistry , Receptor, ErbB-2/immunology , Acylation , Alkynes/chemistry , Antibodies, Monoclonal/immunology , Azides/chemistry , Click Chemistry , Cycloaddition Reaction , Fluorescent Dyes/chemistry , Humans , Immunoconjugates/immunology , Molecular Structure , Oligonucleotides/chemistry , Succinimides/chemistry , Toxins, Biological/chemistry
The vast majority of antibody-drug conjugates (ADC) are prepared through amine-to-thiol conjugation. To date, N-Succinimidyl-4-(maleimidomethyl) cyclohexanecarboxylate (SMCC) has been one of the most frequently applied reagents for the preparation of ADC and other functional conjugates. However, SMCC-based conjugates suffer from limited stability in blood circulation and from a hydrophobic character of the linker, which may give rise to major pharmacokinetic implications. To address this issue, we have developed a heterobifunctional analogue of a SMCC reagent, i.e., sodium 4-(maleimidomethyl)-1,3-dioxane-5-carbonyl)oxy)-2,3,5,6- tetrafluorobenzenesulfonate (MDTF) for amine-to-thiol conjugation. By replacing the cyclohexyl ring in the SMCC structure with the 1,3-dioxane, we increased the hydrophilicity of the linker. A FRET probe based on MD linker was prepared and showed superior stability compared to the MCC linker in human plasma, as well as in a variety of aqueous buffers. A detailed investigation demonstrated an accelerated succinimide ring opening for MD linker, resulting in stabilized conjugates. Finally, the MDTF reagent was applied for the preparation of serum stable antibody-dye conjugate.
