RESUMEN
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 inâ vitro.
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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.
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Inmunoconjugados , Proteínas , Proteínas/química , Lisina/química , Aminoácidos , Anticuerpos , Fenómenos QuímicosRESUMEN
Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.
Asunto(s)
Proteína BRCA1/deficiencia , Dineínas Citoplasmáticas/metabolismo , ADN/metabolismo , Genes BRCA1 , Proteína Homóloga de MRE11/metabolismo , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Aberraciones Cromosómicas , Daño del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Edición Génica , Inestabilidad Genómica/efectos de los fármacos , Recombinación Homóloga/efectos de los fármacos , Humanos , Mutación , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Platino (Metal)/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Unión Proteica , Reparación del ADN por Recombinación/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
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.
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Neoplasias , Hipoxia Tumoral , Humanos , Ratones , Animales , Carotenoides , Neoplasias/terapia , Vitamina A/uso terapéuticoRESUMEN
P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.
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Proteínas Portadoras/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sitios de Unión , Roturas del ADN de Doble Cadena , Reparación del ADN , Femenino , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Fosforilación , Unión Proteica , Dominios Proteicos , Proteínas de Unión al ARN , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/químicaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.
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Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Inhibidores de Proteasoma/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Humanos , Ratones , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Proteasoma/química , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Human pancreatic ductal adenocarcinoma (PDAC) contains a distinctively dense stroma that limits the accessibility of anticancer drugs, contributing to its poor overall prognosis. Nanoparticles can enhance drug delivery and retention in pancreatic tumors and have been utilized clinically for their treatment. In preclinical studies, various mouse models differentially recapitulate the microenvironmental features of human PDAC. Here, we demonstrate that through utilization of different organic cosolvents and by doping of a homopolymer of poly(ε-caprolactone), a diblock copolymer composition of poly(ethylene oxide)- block-poly(ε-caprolactone) may be utilized to generate biodegradable and nanoscale micelles with different physical properties. Noninvasive optical imaging was employed to examine the pharmacology and biodistribution of these various nanoparticle formulations in both allografted and autochthonous mouse models of PDAC. In contrast to the results reported with transplanted tumors, spherical micelles as large as 300 nm in diameter were found to extravasate in the autochthonous model, reaching a distance of approximately 20 µm from the nearest tumor cell clusters. A lipophilic platinum(IV) prodrug of oxaliplatin was further able to achieve a â¼7-fold higher peak accumulation and a â¼50-fold increase in its retention half-life in pancreatic tumors when delivered with 100 nm long worm-like micelles as when compared to the free drug formulation of oxaliplatin. Through further engineering of nanoparticle properties, as well as by widespread adoption of the autochthonous tumor model for preclinical testing, future therapeutic formulations may further enhance the targeting and penetration of anticancer agents to improve survival outcomes in PDAC.
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Carcinoma Ductal Pancreático/diagnóstico por imagen , Lactonas/análisis , Nanopartículas/análisis , Trasplante de Neoplasias/diagnóstico por imagen , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Pancreáticas/diagnóstico por imagen , Polietilenglicoles/análisis , Animales , Antineoplásicos/administración & dosificación , Carcinoma Ductal Pancreático/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Lactonas/farmacocinética , Ratones , Ratones Desnudos , Micelas , Neoplasias Experimentales/tratamiento farmacológico , Imagen Óptica/métodos , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Polietilenglicoles/farmacocinéticaRESUMEN
Selective killing of cancer cells while minimizing damage to healthy tissues is the goal of clinical radiation therapy. This therapeutic ratio can be improved by image-guided radiation delivery and selective radiosensitization of cancer cells. Here, we have designed and tested a novel trimodal theranostic nanoparticle made of bismuth and gadolinium for on-site radiosensitization and image contrast enhancement to improve the efficacy and accuracy of radiation therapy. We demonstrate in vivo magnetic resonance (MR), computed tomography (CT) contrast enhancement, and tumor suppression with prolonged survival in a non-small cell lung carcinoma model during clinical radiation therapy. Histological studies show minimal off-target toxicities due to the nanoparticles or radiation. By mimicking existing clinical workflows, we show that the bismuth-gadolinium nanoparticles are highly compatible with current CT-guided radiation therapy and emerging MR-guided approaches. This study reports the first in vivo proof-of-principle for image-guided radiation therapy with a new class of theranostic nanoparticles.
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Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/radioterapia , Bismuto/uso terapéutico , Medios de Contraste/uso terapéutico , Gadolinio/uso terapéutico , Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/radioterapia , Nanopartículas/uso terapéutico , Dióxido de Silicio/uso terapéutico , Células A549 , Adenocarcinoma del Pulmón , Animales , Bismuto/química , Medios de Contraste/química , Gadolinio/química , Humanos , Imagen por Resonancia Magnética , Ratones , Nanopartículas/química , Radioterapia Guiada por Imagen , Dióxido de Silicio/química , Nanomedicina Teranóstica , Tomografía Computarizada por Rayos XRESUMEN
More than 50% of all cancer patients receive radiation therapy. The clinical delivery of curative radiation dose is strictly restricted by the proximal healthy tissues. We propose a dual-targeting strategy using vessel-targeted-radiosensitizing gold nanoparticles and conformal-image guided radiation therapy to specifically amplify damage in the tumor neoendothelium. The resulting tumor vascular disruption substantially improved the therapeutic outcome and subsidized the radiation/nanoparticle toxicity, extending its utility to intransigent or nonresectable tumors that barely respond to standard therapies.
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Oro/efectos adversos , Nanopartículas del Metal/efectos adversos , Neoplasias/radioterapia , Neovascularización Patológica/tratamiento farmacológico , Línea Celular Tumoral , Endotelio/efectos de los fármacos , Endotelio/patología , Endotelio/efectos de la radiación , Oro/química , Humanos , Nanopartículas del Metal/administración & dosificación , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica/patología , Neovascularización Patológica/radioterapia , Tolerancia a Radiación/efectos de los fármacos , Radioterapia Guiada por ImagenRESUMEN
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 VHHs 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 VHHs 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.
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Nanopartículas , Péptidos , Nanopartículas/química , Animales , Humanos , Ratones , Péptidos/química , Línea Celular Tumoral , Femenino , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Neoplasias de la Mama/metabolismoRESUMEN
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.
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Anticuerpos Monoclonales , Quitosano , Humanos , Ratones , Animales , Anticuerpos Monoclonales/farmacocinética , Hidrogeles , Preparaciones de Acción Retardada , Inyecciones SubcutáneasRESUMEN
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.
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Anticuerpos Monoclonales , Farmacia , Humanos , Anticuerpos Monoclonales/química , Fenómenos Mecánicos , Transportes/métodosRESUMEN
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.
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Mieloma Múltiple , Profármacos , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Bortezomib/uso terapéutico , Dexametasona/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologíaRESUMEN
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.
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Cromatina , Histonas , Proteína 1 de Unión al Supresor Tumoral P53 , Roturas del ADN de Doble Cadena , Reparación del ADN , Histonas/metabolismo , Ingeniería de Proteínas , Proteína 1 de Unión al Supresor Tumoral P53/antagonistas & inhibidores , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , HumanosRESUMEN
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.
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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.