Rabbit derived VL single-domains as promising scaffolds to generate antibody–drug conjugates

© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Antibody-drug conjugates (ADCs) are among the fastest-growing classes of therapeutics in oncology. Although ADCs are in the spotlight, they still present significant engineering challenges. Therefore, there is an urgent need to develop more stable and effective ADCs. Most rabbit light chains have an extra disulfide bridge, that links the variable and constant domains, between Cys80 and Cys171, which is not found in the human or mouse. Thus, to develop a new generation of ADCs, we explored the potential of rabbit-derived VL-single-domain antibody scaffolds (sdAbs) to selectively conjugate a payload to Cys80. Hence, a rabbit sdAb library directed towards canine non-Hodgkin lymphoma (cNHL) was subjected to in vitro and in vivo phage display. This allowed the identification of several highly specific VL-sdAbs, including C5, which specifically target cNHL cells in vitro and present promising in vivo tumor uptake. C5 was selected for SN-38 site-selective payload conjugation through its exposed free Cys80 to generate a stable and homogenous C5-DAB-SN-38. C5-DAB-SN-38 exhibited potent cytotoxicity activity against cNHL cells while inhibiting DNA-TopoI activity. Overall, our strategy validates a platform to develop a novel class of ADCs that combines the benefits of rabbit VL-sdAb scaffolds and the canine lymphoma model as a powerful framework for clinically translation of novel therapeutics for cancer.This work was supported by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP (SAICT/2017/32085, PTDC/QUI-OUT/3989/2021 and Ph.D. fellowship SFRH/BD/131468/2017 to ASA and SFRH/BD/90514/2012 to JD). CIISA has provided support through Project UIDB/00276/2020, funded by FCT and LA/P/0059/2020-AL4AnimalS. Research Institute for Medicines (iMed.ULisboa) acknowledges the financial support of Fundação para a Ciência e Tecnologia (Projects: PTDC/QUI-OUT/3989/2021; UIDB/04138/2020 and UIDP/04138/2020). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).info:eu-repo/semantics/publishedVersio

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

Inserção C-H de carbenóides de ródio em água e reutilização do catalisador Rhodium (II) carbene C-H insertion in water and catalyst reuse

<abstract language="eng">A five-session laboratory experiment is described for the synthesis of a beta-lactam via Rh(II) catalysed intramolecular C-H insertion of a alpha-diazo-alpha-ethoxycarbonylacetamide. The metallo-carbene, responsible for the C-H bond activation, was generated from the diazo substrate and the catalyst Rh2(OAc)4. The high stability and solubility of the catalyst and the exclusive C-H insertion of the Rh-carbene allows the synthesis of this important heterocycle in water and the catalyst reutilization

C-H carbene insertion of alpha-diazo acetamides by photolysis in non-conventional media

Light from a mercury vapor high-pressure lamp was used to induce the photolytic decomposition of alpha-diazo acetamides in hexane and in nonconventional media such as water or a film. The corresponding beta- and/or gamma-lactams were obtained in reasonable yields and in some cases with good diastereoselectivities with no need to use a metallic catalyst. Experimental studies on chiral substrates demonstrated the occurrence of insertion with retention of configuration

<i>N</i>‑Terminal Cysteine Bioconjugation with (2-Cyanamidophenyl)boronic Acids Enables the Direct Formation of Benzodiazaborines on Peptides

Benzodiazaborines (BDABs) have emerged as a valuable tool to produce stable and functional bioconjugates via a click-type transformation. However, the current available methods to install them on peptides lack bioorthogonality, limiting their applications. Here, we report a strategy to install BDABs directly on peptide chains using (2-cyanamidophenyl)boronic acids (2CyPBAs). The resulting BDAB is stabilized through the formation of a key intramolecular B–N bond. This technology was applied in the selective modification of N-terminal cysteine-containing functional peptides

Tetra-mu-acetato-bis{[1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene]rhodium(II)}(Rh-Rh) tetrahydrofuran tetrasolvate

In this paper, we compare and discuss the very different crystal structures and supramolecular arrangements obtained when using different crystallization solvents with the same organometallic moiety. The new title tetrahydrofuran (THF) solvate, [Rh-2(C2H3O2)(4)(C27H36N2)(2)]center dot 4C(4)H(8)O, is compared with the toluene trisolvate reported previously by us [Gois, Trindade, Veiros, Andre, Duarte, Afonso, Caddick & Cloke (2007). Angew. Chem. Int. Ed. 46, 5750-.5753]. The molecular structures of the two complex molecules display a similar conformation, but due to the presence of different solvent molecules, the two solvates crystallize in different space groups and exhibit quite diverse supramolecular assemblies. The toluene solvate crystallizes in the triclinic space group P (1) over bar, while in the presence of THF, the monoclinic P2(1)/c space group is obtained, with the complex molecule residing on an inversion centre. The resulting crystal packing displays no classical hydrogen bonds but different supramolecular synthons give rise to different packing motifs. In this work, we highlight the different supramolecular architectures obtained when organometallic moieties crystallize with different solvent molecules. We compare the novel structure of the THF derivative with that of the toluene solvate of a dirhodium(II) complex belonging to a new family of catalyst compounds exhibiting very high performance in arylation processes.. - Fundacao para a Ciencia e Tecnologia [POCI2010]; FEDER [PTDC/QUI/66695/2006, PTDC/QUI/66015/2006, POCI/QUI/60175/2004, POCI/QUI/58791/2004, SFRH/BPD/18624/2004, SFRH/BD/30619/2006, SFRH/BD/40474/2007]. - The authors acknowledge funding of the project by Fundacao para a Ciencia e Tecnologia (grant No. POCI2010) and FEDER (grant Nos. PTDC/QUI/66695/2006, PTDC/QUI/66015/2006, POCI/QUI/60175/2004, POCI/QUI/58791/2004, SFRH/BPD/18624/2004, SFRH/BD/30619/2006 and SFRH/BD/40474/2007

Cysteine‐Assisted Click‐Chemistry for Proximity‐Driven, Site‐Specific Acetylation of Histones

Abstract: Post‐translational modifications of histones are essential in the regulation of chromatin structure and function. Among these modifications, lysine acetylation is one of the most established. Earlier studies relied on the use of chromatin containing heterogeneous mixtures of histones acetylated at multiple sites. Differentiating the individual contribution of single acetylation events towards chromatin regulation is thus of great relevance. However, it is difficult to access homogeneous samples of histones, with a single acetylation, in sufficient quantities for such studies. By engineering histone H3 with a cysteine in proximity of the lysine of interest, we demonstrate that conjugation with maleimide‐DBCO followed by a strain‐promoted alkyne‐azide cycloaddition reaction results in the acetylation of a single lysine in a controlled, site‐specific manner. The chemical precision offered by our click‐to‐acetylate approach will facilitate access to and the study of acetylated histones