Chemical Production of Cytotoxic Bispecific Antibodies Using the Ugi Multicomponent Reaction.

Bispecific antibodies (bsAbs) have recently emerged as a promising platform for the treatment of several conditions, most importantly cancer. Based on the combination of two different antigen-binding motifs in a single macromolecule; bsAbs can either display the combined characteristics of their parent antibodies, or new therapeutic features, inaccessible by the sole combination of two distinct antibodies. While bsAbs are traditionally produced by molecular biology techniques, the chemical development of bsAbs holds great promises and strategies have just begun to surface. In this context, we took advantage of a chemical strategy based on the use of the Ugi reaction for the site-selective conjugation of whole antibodies and coupled the resulting conjugates in a bioorthogonal manner with Fab fragments, derived from various antibodies. We thus managed to produce five different bsAbs with 2 : 1 valency, with yields ranging from 20 % to 48 %, and showed that the affinity of the parent antibody was preserved in all bsAbs. We further demonstrated the interest of our strategy by producing two other bsAbs behaving as cytotoxic T cell engagers with IC50 values in the picomolar range in vitro.

Targeted delivery of immune-stimulating bispecific RNA, inducing apoptosis and anti-tumor immunity in cancer cells.

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.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

Subcutaneous Administration of a Zwitterionic Chitosan-Based Hydrogel for Controlled Spatiotemporal Release of Monoclonal Antibodies.

Subcutaneous (SC) administration of monoclonal antibodies (mAbs) is a proven strategy for improving therapeutic outcomes and patient compliance. The current FDA-/EMA-approved enzymatic approach, utilizing recombinant human hyaluronidase (rHuPH20) to enhance mAbs SC delivery, involves degrading the extracellular matrix's hyaluronate to increase tissue permeability. However, this method lacks tunable release properties, requiring individual optimization for each mAb. Seeking alternatives, physical polysaccharide hydrogels emerge as promising candidates due to their tunable physicochemical and biodegradability features. Unfortunately, none have demonstrated simultaneous biocompatibility, biodegradability, and controlled release properties for large proteins (≥150 kDa) after SC delivery in clinical settings. Here, a novel two-component hydrogel comprising chitosan and chitosan@DOTAGA is introduced that can be seamlessly mixed with sterile mAbs formulations initially designed for intravenous (IV) administration, repurposing them as novel tunable SC formulations. Validated in mice and nonhuman primates (NHPs) with various mAbs, including trastuzumab and rituximab, the hydrogel exhibited biodegradability and biocompatibility features. Pharmacokinetic studies in both species demonstrated tunable controlled release, surpassing the capabilities of rHuPH20, with comparable parameters to the rHuPH20+mAbs formulation. These findings signify the potential for rapid translation to human applications, opening avenues for the clinical development of this novel SC biosimilar formulation.