INA03, a potent transferrin-competitive antibody-drug conjugate against CD71, for a safer acute leukemia treatment.

Innovative strategies to enhance efficacy and overcome drug resistance in hematologic cancers such as antibody-drug conjugates (ADCs) have shifted the paradigm of conventional care by delivering promising outcomes in cancer therapies with a significant reduction in the risk of relapse. The transferrin receptor 1, CD71, known to be overexpressed in malignant cells, is considered a potent anti-tumoral target. Therefore, we have developed an anti-CD71 ADC, INA03, a humanized antibody conjugated to the monomethyl auristatin E (MMAE) through a 3-arylpropiolonitrile-valine-citruline linker. In this study, we investigated both potency and safety of INA03, in competition with transferrin (Tf), the CD71's natural ligand, as a novel strategy to specifically target highly proliferative cells. The high expression of CD71 was confirmed on different leukemic cell lines, allowing INA03 to bind efficiently. Subsequently, INA03 rapidly internalizes into lysosomal compartments, where its cytotoxic drug is released following cathepsin-B cleavage. Downregulating CD71 expression using shRNA highlighted that INA03-induced cell death was dependent on CD71 density at the cell surface. INA03 intravenous treatment in acute leukemia mouse models significantly reduced tumor burden, increased mice survival and showed no residual disease compared to conventional chemotherapies. Since INA03 competes with the human Tf, a double knock-in (hCD71/hTf) competent mouse model was generated to mimic human pharmacokinetics and pharmacodynamics. INA03 administration in hCD71/hTf mice did not reveal, even at high doses, any improper toxicities. Hence, these data demonstrate promising pre-clinical efficacy and safety of INA03 and support its development as a novel acute leukemia treatment.

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, 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.

Thermofluorimetric Analysis (TFA) using Probes with Flexible Spacers: Application to Direct Antibody Sensing and to Antibody-Oligonucleotide (AbO) Conjugate Valency Monitoring.

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.

Non-specific interactions of antibody-oligonucleotide conjugates with living cells.

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.

On the use of DNA as a linker in antibody-drug conjugates: synthesis, stability and in vitro potency.

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.

Automated linkage of proteins and payloads producing monodisperse conjugates.

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 as Therapeutic, Imaging, and Detection Agents.

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.

Reduction-rebridging strategy for the preparation of ADPN-based antibody-drug conjugates.

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.

Design and Synthesis of Iminosydnones for Fast Click and Release Reactions with Cycloalkynes.

Emerging applications in the field of chemical biology are currently limited by the lack of bioorthogonal reactions allowing both removal and linkage of chemical entities on complex biomolecules. We recently discovered a novel reaction between iminosydnones and strained alkynes leading to two products resulting from ligation and fragmentation of iminosydnones under physiological conditions. We now report the synthesis of a panel of substituted iminosydnones and the structure reactivity relationship between these compounds and strained alkyne partners. This study identified the most relevant substituents, which allow to increase the rate of the transformation and to develop a bifunctional cleavable linker with improved kinetics.

Bioorthogonal Click and Release Reaction of Iminosydnones with Cycloalkynes.

We report the discovery of a new bioorthogonal click-and-release reaction involving iminosydnones and strained alkynes. This transformation leads to two products resulting from the ligation and fragmentation of iminosydnones under physiological conditions. Optimized iminosydnones were successfully used to design innovative cleavable linkers for protein modification, thus opening up new areas in the fields of drug release and target-fishing applications. This click-and-release technology offers the possibility of exchanging tags on proteins for functionalized cyclooctynes under mild and bioorthogonal conditions.

Development and evaluation of ß-galactosidase-sensitive antibody-drug conjugates.

The selective destruction of tumour cells while sparing healthy tissues is one of the main challenges in cancer therapy. Antibody-drug conjugates (ADCs) are arguably the most rapidly expanding class of targeted cancer therapies. Efficient drug conjugation and release technologies are essential for the development of these new therapeutic agents. In response to the ever-increasing demand for efficient drug release systems, we have developed a new class of ß-galactosidase-cleavable linkers for ADCs. Within this framework, novel payloads comprising a galactoside linker, the monomethyl auristatin E (MMAE) and cysteine-reactive groups were synthesized, conjugated with trastuzumab and evaluated both in vitro and in vivo. The ADCs with galactoside linkers demonstrated superior therapeutic efficacy in mice compared to the marketed trastuzumab emtansine used for the treatment of breast cancer.

Targeting the tumour microenvironment with an enzyme-responsive drug delivery system for the efficient therapy of breast and pancreatic cancers.

The development of novel therapeutic strategies allowing the destruction of tumour cells while sparing healthy tissues is one of the main challenges of cancer chemotherapy. Here, we report on the design and antitumour activity of a low-molecular-weight drug delivery system programmed for the selective release of the potent monomethylauristatin E in the tumour microenvironment of solid tumours. After intravenous administration, this compound binds covalently to plasmatic albumin through Michael addition, thereby enabling its passive accumulation in tumours where extracellular ß-glucuronidase initiates the selective release of the drug. This targeting device produces outstanding therapeutic efficacy on orthotopic triple-negative mammary and pancreatic tumours in mice (50% and 33% of mice with the respective tumours cured), leading to impressive reduction or even disappearance of tumours without inducing side effects.

Acyl Fluorides: Fast, Efficient, and Versatile Lysine-Based Protein Conjugation via Plug-and-Play Strategy.

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.

2-(Maleimidomethyl)-1,3-Dioxanes (MD): a Serum-Stable Self-hydrolysable Hydrophilic Alternative to Classical Maleimide Conjugation.

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.

Ultrafast Click Chemistry with Fluorosydnones.

We report the synthesis and reactivity of 4-fluorosydnones, a unique class of mesoionic dipoles displaying exquisite reactivity towards both copper-catalyzed and strain-promoted cycloaddition reactions with alkynes. Synthetic access to these new mesoionic compounds was granted by electrophilic fluorination of σ-sydnone Pd(II) precursors in the presence of Selectfluor. Their reactions with terminal and cyclic alkynes were found to proceed very rapidly and selectively, affording 5-fluoro-1,4-pyrazoles with bimolecular rate constants up to 10(4) m(-1) s(-1) , surpassing those documented in the literature with cycloalkynes. Kinetic studies were carried out to unravel the mechanism of the reaction, and the value of 4-fluorosydnones was further highlighted by successful radiolabeling with [(18) F]Selectfluor.

Palladium-Catalyzed Chemoselective and Biocompatible Functionalization of Cysteine-Containing Molecules at Room Temperature.

The third generation of aminobiphenyl palladacycle pre-catalyst "G3-Xantphos" enables functionalization of peptides containing cysteine in high yields. The conjugation (bioconjugation) occurs chemoselectively at room temperature under biocompatible conditions. Extension of the method to protein functionalization allows selective bioconjugation of the trastuzumab antibody.

MAPN: First-in-Class Reagent for Kinetically Resolved Thiol-to-Thiol Conjugation.

Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)-an efficient reagent for kinetically resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide, MAPN has shown substantial advantages for the preparation of thiol-thiol heteroconjugates. Namely, an antibody-drug conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.

CBTF: new amine-to-thiol coupling reagent for preparation of antibody conjugates with increased plasma stability.

Amine-to-thiol coupling is the most common route for the preparation of antibody-drug conjugates (ADC). It is usually achieved by using heterobifunctional reagents possessing an activated ester at one end and a maleimide group at the other. However, maleimide-based conjugates were recently revealed to have limited stability in blood circulation, which can compromise therapeutic efficacy of the conjugate. To address this issue, we have developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling. It comprises a recently described 3-arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. A series of antibody-dye conjugates have been prepared using this reagent and shown superior stability in human blood plasma compared to maleimide-derived conjugates.

Copper(I)-catalyzed cycloaddition of 4-bromosydnones and alkynes for the regioselective synthesis of 1,4,5-trisubstituted pyrazoles.

Copper-catalyzed cycloaddition of alkynes with 4-bromosydnones provides a convenient, mild, and regioselective method for the synthesis of a wide range of bromopyrazoles. The broad functional group tolerance of the cycloaddition reaction and further palladium-catalyzed cross-coupling reactions allowed the preparation of polyfunctionalized 1,4,5-pyrazoles that are otherwise difficult to obtain by conventional methods.