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.

Supramolecular Heterodimer Peptides Assembly for Nanoparticles Functionalization.

Nanoparticle (NP) surface functionalization with proteins, including monoclonal antibodies (mAbs), mAb fragments, and various peptides, has emerged as a promising strategy to enhance tumor targeting specificity and immune cell interaction. However, these methods often rely on complex chemistry and suffer from batch-dependent outcomes, primarily due to limited control over the protein orientation and quantity on NP surfaces. To address these challenges, a novel approach based on the supramolecular assembly of two peptides is presented to create a heterotetramer displaying VH Hs on NP surfaces. This approach effectively targets both tumor-associated antigens (TAAs) and immune cell-associated antigens. In vitro experiments showcase its versatility, as various NP types are biofunctionalized, including liposomes, PLGA NPs, and ultrasmall silica-based NPs, and the VH Hs targeting of known TAAs (HER2 for breast cancer, CD38 for multiple myeloma), and an immune cell antigen (NKG2D for natural killer (NK) cells) is evaluated. In in vivo studies using a HER2+ breast cancer mouse model, the approach demonstrates enhanced tumor uptake, retention, and penetration compared to the behavior of nontargeted analogs, affirming its potential for diverse applications.

Site-Selective Protein Conjugation by a Multicomponent Ugi Reaction.

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.

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.

Influence of pneumatic transportation on the stability of monoclonal antibodies.

Pneumatic transportation systems (PTS) were recently proposed as a method to carry ready-for-injection diluted monoclonal antibodies (mAbs) from the pharmacy to the bedside of patients. This method reduces transportation time and improves the efficiency of drug distribution process. However, mAbs are highly sensitive molecules for which subtle alterations may lead to deleterious clinical effects. These alterations can be caused by various external factors such as temperature, pH, pressure, and mechanical forces that may occur during transportation. Hence, it is essential to ensure that the mAbs transported by PTS remain stable and active throughout the transportation process. This study aims to determine the safety profile of PTS to transport 11 routinely used mAbs in a clinical setting through assessment of critical quality attributes (CQA) and orthogonal analysis. Hence, we performed aggregation/degradation profiling, post-translational modifications identification using complementary mass spectrometry-based methods, along with visible and subvisible particle formation determination by light absorbance and light obscuration analysis. Altogether, these results highlight that PTS can be safely used for this purpose when air is removed from the bags during preparation.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1-autoinhibited for chromatin binding-that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1 - autoinhibited for chromatin binding - that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

Clinically Translatable Transcrocetin Delivery Platform for Correction of Tumor Hypoxia and Enhancement of Radiation Therapy Effects.

Improving the tumor reoxygenation to sensitize the tumor to radiation therapy is a cornerstone in radiation oncology. Here, the pre-clinical development of a clinically transferable liposomal formulation encapsulating trans sodium crocetinate (NP TSC) is reported to improve oxygen diffusion through the tumor environment. Early pharmacokinetic analysis of the clinical trial of this molecule performed on 37 patients orient to define the optimal fixed dosage to use in a triple-negative breast cancer model to validate the therapeutic combination of radiation therapy and NP TSC. Notably, it is reported that this formulation is non-toxic in both humans and mice at the defined fixed concentration, provides a normalization of the tumor vasculature within 72 h window after systemic injection, leads to a transient increase (50% improvement) in the tumor oxygenation, and significantly improves the efficacy of both mono-fractionated and fractionated radiation therapy treatment. Together, these findings support the introduction of a first-in-class therapeutic construct capable of tumor-specific reoxygenation without associated toxicities.

Molecular bottlebrush prodrugs as mono- and triplex combination therapies for multiple myeloma.

Cancer therapies often have narrow therapeutic indexes and involve potentially suboptimal combinations due to the dissimilar physical properties of drug molecules. Nanomedicine platforms could address these challenges, but it remains unclear whether synergistic free-drug ratios translate to nanocarriers and whether nanocarriers with multiple drugs outperform mixtures of single-drug nanocarriers at the same dose. Here we report a bottlebrush prodrug (BPD) platform designed to answer these questions in the context of multiple myeloma therapy. We show that proteasome inhibitor (bortezomib)-based BPD monotherapy slows tumour progression in vivo and that mixtures of bortezomib, pomalidomide and dexamethasone BPDs exhibit in vitro synergistic, additive or antagonistic patterns distinct from their corresponding free-drug counterparts. BPDs carrying a statistical mixture of three drugs in a synergistic ratio outperform the free-drug combination at the same ratio as well as a mixture of single-drug BPDs in the same ratio. Our results address unanswered questions in the field of nanomedicine, offering design principles for combination nanomedicines and strategies for improving current front-line monotherapies and combination therapies for multiple myeloma.

A Combination of Native LC-MS Approaches for the Comprehensive Characterization of the Antibody-Drug Conjugate Trastuzumab Deruxtecan.

BACKGROUND: Native mass spectrometry (nMS) approaches appear attractive to complement bottom-up strategies traditionally used in biopharmaceutical industries thanks to their quite straightforward and rapid workflows, especially through online hyphenation of non-denaturing liquid chromatography (LC) to nMS. The present work provides an overview of the state-of-the-art chromatographic tools available for the detailed characterization of monoclonal antibody (mAb) formats, exemplified on the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd). METHODS: T-DXd was first characterized by conventional reversed phase LC (rpLC) and peptide mapping. Couplings of size exclusion chromatography (SEC), cation exchange chromatography (CEX), and hydrophobic interaction chromatography (HIC) to nMS were used to gain further insights into size, hydrophobic, and charge variants of T-DXd and its parental mAb trastuzumab, at intact and middle-up levels. RESULTS: SEC-nMS first offered a direct snapshot of the homogeneous conjugation of T-DXd, with an average drug-to-antibody ratio (DAR) of 8 in agreement with a conjugation on cysteines after reduction of all interchain disulfide bonds. Moreover, SEC-nMS afforded precise identification and quantification of aggregates and fragments. Middle-up level experiments performed after IdeS digestion confirmed that drug conjugation occurs in the Fab region of the mAb, as seen with rpLC. HIC separated two DAR8 species that could not be differentiated by nMS. Although middle-up HIC-nMS proved to be more informative for oxidized forms, the identification of minor variants was still difficult because of poor MS signal quality, showing how the coupling of HIC to nMS remains challenging. Lastly, middle-up CEX-nMS provided accurate determination and localization of post-translational modifications, with several acidic/basic variants within Fab and Fc regions of T-DXd that were also identified by peptide mapping. CONCLUSIONS: This study illustrates the strengths and drawbacks of each LC-nMS coupling. By combining SEC-, HIC-, and CEX-nMS, we were able to achieve a comprehensive characterization of T-DXd without extensive sample preparation prior to MS analysis.

Optimal Physicochemical Properties of Antibody-Nanoparticle Conjugates for Improved Tumor Targeting.

Tumor-targeted antibody (mAb)/fragment-conjugated nanoparticles (NPs) represent an innovative strategy for improving the local delivery of small molecules. However, the physicochemical properties of full mAb-NPs and fragment-NPs-that is, NP material, size, charge, as well as the targeting antibody moiety, and the linker conjugation strategies-remain to be optimized to achieve an efficient tumor targeting. A meta-analysis of 161 peer-reviewed studies is presented, which describes the use of tumor-targeted mAb-NPs and fragment-NPs from 2009 to 2021. The use of these targeted NPs is confirmed to result in significantly greater tumor uptake of NPs than that of naked NPs (7.9 ± 1.9% ID g-1 versus 3.2 ± 0.6% ID g-1 , respectively). The study further demonstrates that for lipidic NPs, fragment-NPs provide a significantly higher tumor uptake than full mAb-NPs. In parallel, for both polymeric and organic/inorganic NPs, full mAb-NPs yield a significant higher tumor uptake than fragment-NPs. In addition, for both lipidic and polymeric NPs, the tumor uptake is improved with the smallest sizes of the conjugates. Finally, the pharmacokinetics of the conjugates are demonstrated to be driven by the NPs and not by the antibody moieties, independently of using full mAb-NPs or fragment-NPs, confirming the importance of optimizing the NP design to improve the tumor uptake.

Anti-BCMA Immuno-NanoPET Radiotracers for Improved Detection of Multiple Myeloma.

Current clinical imaging modalities for the sensitive and specific detection of multiple myeloma (MM) rely on nonspecific imaging contrast agents based on gadolinium chelates for magnetic resonance imaging (MRI) or for 18 F-FDG-directed and combined positron emission tomography (PET) and computed tomography (CT) scans. These tracers are not, however, able to detect minute plasma cell populations in the tumor niche, leading to false negative results. Here, a novel PET-based anti-BCMA nanoplatform labeled with 64 Cu is developed to improve the monitoring of these cells in both the spine and femur and to compare its sensitivity and specificity to more conventional immunoPET (64 Cu labeled anti-BCMA antibody) and passively targeted PET radiotracers (64 CuCl2 and 18 F-FDG). This proof-of-concept preclinical study confirmed that by conjugating up to four times more radioisotopes per antibody with the immuno-nanoPET platform, an improvement in the sensitivity and in the specificity of PET to detect tumor cells in an orthotopic model of MM is observed when compared to the traditional immunoPET approach. It is anticipated that when combined with tumor biopsy, this immuno-nanoPET platform may improve the management of patients with MM.

TROIKA-1: A double-blind, randomized, parallel group, study aimed to demonstrate the equivalent pharmacokinetic profile of HD201, a potential biosimilar candidate to trastuzumab, versus EU-Herceptin® and US-Herceptin® in healthy male subjects.

Prestige Biopharma Ltd (Singapore) has developed HD201, a proposed biosimilar to reference product trastuzumab. As a part of the stepwise approach to ensure comparability between the biosimilar candidate and the reference medicinal product, a phase I study in healthy subjects was conducted to demonstrate the pharmacokinetic (PK) equivalence (NCT03776240). The primary objective of the study was to demonstrate (PK) equivalence of HD201, EU-Herceptin® , and US-Herceptin® given at 6 mg/kg as a 90-min i.v. infusion to healthy male subjects. A pairwise comparisons based on the primary endpoint AUC0-inf and secondary PK endpoints, AUC0-last and Cmax were undertaken. PK equivalence was to be concluded if the 90% confidence interval (CI) for the ratio of geometric means for each criterion were within the equivalence margin of 80% to 125%. Secondary objectives included assessment of other PK parameters, safety, tolerability, and immunogenicity in the three arms. A total of 105 healthy male subjects (35/treatment) were randomized in this study. The 90% CI for the ratios of AUC0-inf , Cmax and AUC0-last , were within 80%-125% for the comparisons of HD201 to EU-Herceptin® or US-Herceptin® and EU-Herceptin® to US-Herceptin® . The frequency of subjects with TEAEs of special interest was slightly lower in the HD201 group (20.0%) compared to the other treatment groups (EU-Herceptin® : 34.3%; US-Herceptin® : 31.4%). Only 1 subject (EU-Herceptin® group) developed anti-drug antibodies prior to dosing. Overall, HD201 demonstrates PK similarity to both EU-Herceptin® and US-Herceptin® . The three study drugs also demonstrated similar safety profiles.

Liposomal encapsulation of trans-crocetin enhances oxygenation in patients with COVID-19-related ARDS receiving mechanical ventilation.

Current therapeutic treatments improving the impaired transportation of oxygen in acute respiratory distress syndrome (ARDS) have been found to be relevant and beneficial for the therapeutic treatment of COVID-19 patients suffering from severe respiratory complications. Hence, we report the preclinical and the preliminary results of the Phase I/II clinical trial of LEAF-4L6715, a liposomal nanocarrier encapsulating the kosmotropic agent trans-crocetin (TC), which, once injected, enhance the oxygenation of vascular tissue and therefore has the potential to improve the clinical outcomes of ARDS and COVID-19 in severely impacted patients. We demonstrated that the liposomal formulation enabled to increase from 30 min to 48 h the reoxygenation properties of free TCs in vitro in endothelial cells, but also to improve the half-life of TC by 6-fold in healthy mice. Furthermore, we identified 25 mg/kg as the maximum tolerated dose in mice. This determined concentration led to the validation of the therapeutic efficacy of LEAF-4 L6715 in a sepsis mouse model. Finally, we report the preliminary outcomes of an open-label multicenter Phase I/II clinical trial (EudraCT 2020-001393-30; NCT04378920), which was aimed to define the appropriate schedule and dosage of LEAF-4L6715 and to confirm its tolerability profile and preliminary clinical activity in COVID-19 patients treated in intensive care unit.

Ral GTPases promote breast cancer metastasis by controlling biogenesis and organ targeting of exosomes.

Cancer extracellular vesicles (EVs) shuttle at distance and fertilize pre-metastatic niches facilitating subsequent seeding by tumor cells. However, the link between EV secretion mechanisms and their capacity to form pre-metastatic niches remains obscure. Using mouse models, we show that GTPases of the Ral family control, through the phospholipase D1, multi-vesicular bodies homeostasis and tune the biogenesis and secretion of pro-metastatic EVs. Importantly, EVs from RalA or RalB depleted cells have limited organotropic capacities in vivoand are less efficient in promoting metastasis. RalA and RalB reduce the EV levels of the adhesion molecule MCAM/CD146, which favors EV-mediated metastasis by allowing EVs targeting to the lungs. Finally, RalA, RalB, and MCAM/CD146, are factors of poor prognosis in breast cancer patients. Altogether, our study identifies RalGTPases as central molecules linking the mechanisms of EVs secretion and cargo loading to their capacity to disseminate and induce pre-metastatic niches in a CD146-dependent manner.