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Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.
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Subunidades alfa de Proteínas de Ligação ao GTP/farmacologia , Receptores do FSH/agonistas , beta-Arrestina 2/farmacologia , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , CinéticaRESUMO
Antibody-drug conjugates (ADC) are spearheading vectorized chemotherapy against cancer, with 4 FDA-approved ADCs and 79 in clinical trials. However, most ADCs are produced using a stochastic bioconjugation method, target hematological cancers, and are derived from a full immunoglobulin-G (IgG). These factors limit their efficacy, especially against solid tumors which remain difficult to treat. Here we report the site-specific conjugation of a single auristatin derivative onto an engineered anti-HER2 single chain fragment variable (scFv) of the trastuzumab antibody, generating new scFv-drug conjugates (SDC). Two cysteines were judiciously incorporated at the beginning of the scFv hexahistidine tag, in order to allow controlled bioconjugation of a heterobifunctional linker including a second generation maleimide (SGM), either cleavable (for monomethyl auristatin E) or noncleavable (for monomethyl auristatin F). Our data indicated that both SDCs conserved their affinity to HER2 in comparison to the native scFv, and were efficiently able to kill in vitro HER2-positive SK-BR-3 cells at subnanomolar concentrations (EC50 of 0.68 nM and 0.32 nM). No effect was observed on HER2-negative MCF-7 cells. Ours results showed efficient targeting of site-specific SDCs against HER2-positive breast cancer cells. This work represents a first important step in the design of more effective small conjugates, paving the way for future in vivo translation to evaluate their full potential.
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Aminobenzoatos/química , Neoplasias da Mama/tratamento farmacológico , Imunoconjugados/química , Imunoconjugados/farmacologia , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Maleimidas/química , Oligopeptídeos/química , Receptor ErbB-2/efeitos dos fármacos , Anticorpos de Cadeia Única/química , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/uso terapêutico , Fatores Imunológicos/uso terapêutico , Engenharia de Proteínas , Trastuzumab/química , Trastuzumab/imunologiaRESUMO
Biocompatible multifunctional nanomedicines (NMs) are known to be an attractive platform for targeted anticancer theranosis. However, these nanomedicines are of interest only if they efficiently target diseased cells and accumulate in tumors. Here we report the synthesis of a new generation of immunotargeted nanomedicines composed of a superparamagnetic iron oxide nanoparticle (SPION) core, polyethylene glycol coating and the anti-HER2 single chain fragment variable (scFv) of Trastuzumab antibody. We developed two novel bioengineered scFv carrying two cysteines located (i) at the end (4D5.1-cys2) or (ii) at the beginning (4D5.2-cys2) of its hexahistidine tag. The scFv bioconjugation was controlled via heterobifunctional linkers including a second generation maleimide (SGM). Our data indicated that the insertion of cysteines at the beginning of the hexahistidine tag was allowed to obtain nearly 2-fold conjugation efficiency (13 scFv/NP) compared to NMs using classical maleimide. As a result, the NMs-4D5.2 built using the optimal 4D5-cys2 and linkers equipped with SGM showed the enhanced recognition of HER2 in an ELISA format and on the surface of SK-BR-3 breast cancer cells in vitro. Their stability in serum was also significantly improved compared to the NMs-4D5. Our results showed the fundamental importance of the controlled ligand conjugation in the perspective of rational design of NMs with tailored physicochemical and biological properties.
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Antineoplásicos Imunológicos/química , Imunoconjugados/química , Nanopartículas de Magnetita/química , Maleimidas/química , Anticorpos de Cadeia Única/química , Trastuzumab/química , Anticorpos Imobilizados/química , Anticorpos Imobilizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/farmacologia , Maleimidas/farmacologia , Modelos Moleculares , Anticorpos de Cadeia Única/farmacologia , Trastuzumab/farmacologiaRESUMO
Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG12 spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitro cytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitro potency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.
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Antineoplásicos Imunológicos/química , Catepsina B/metabolismo , Imunoconjugados/química , Oligopeptídeos/química , Receptor ErbB-2/metabolismo , Trastuzumab/química , Antineoplásicos Imunológicos/síntese química , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética/métodos , Sistemas de Liberação de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/farmacologia , Neoplasias/tratamento farmacológico , Oligopeptídeos/síntese química , Oligopeptídeos/farmacologia , Trastuzumab/farmacologiaRESUMO
Antibody-drug conjugates, such as brentuximab vedotin (BTXv), are an innovative category of monoclonal antibodies. BTXv is bioconjugated via the chemical reduction of cysteine residues involved in disulfide bonds. Species of BTXv containing zero, two, four, six, or eight vedotin molecules per antibody coexist in the stock solution. We investigated the influence of drug loading on the binding of the antibody to FcRn, a major determinant of antibody pharmacokinetics in humans. We developed a hydrophobic interaction chromatography (HIC) method for separating the different species present in the stock solution of BTXv, and we purified and characterized the collected species before use. We assessed the binding of these different species to FcRn in a cellular assay based on flow cytometry and surface plasmon resonance. HIC separated the different species of BTXv and allowed their collection at adequate levels of purity. Physicochemical characterization showed that species with higher levels of drug loading tended to form more aggregates. FcRn binding assays showed that the most conjugated species, particularly those with saturated loading, interacted more strongly than unconjugated BTXv with the FcRn.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/metabolismo , Brentuximab Vedotin , Cromatografia em Gel , Citometria de Fluxo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/metabolismo , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Trastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and Drug Administration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor 2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitations included heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimal tumor penetration. To address this, different development strategies are oriented towards homogeneous conjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed next generation ADCs, a key parameter to consider is the management of the hydrophobicity associated with the linker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovative branched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE) and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficient generation of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2. Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this study highlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitro cellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficient reduction of tumor size in vivo. These results are very promising and encourage us to explore further fragment-drug conjugate development.
Assuntos
Aminobenzoatos , Neoplasias da Mama , Imunoconjugados , Oligopeptídeos , Estados Unidos , Camundongos , Humanos , Animais , Feminino , Neoplasias da Mama/tratamento farmacológico , Preparações Farmacêuticas , Distribuição Tecidual , Linhagem Celular Tumoral , Imunoconjugados/uso terapêutico , Ado-Trastuzumab Emtansina , Interações Hidrofóbicas e Hidrofílicas , PolietilenoglicóisRESUMO
BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.
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Antibody-drug conjugates (ADCs) are targeted therapies, mainly used in oncology, consisting in a three-component molecular architecture combining a highly potent drug conjugated via a linker onto a monoclonal antibody (mAb), designed for the selective delivery of the drug to the tumor site. The linker is a key component, defining the ADC stability and mechanism of action, and particularly the drug release strategy. In this study, we have developed and synthesized a cleavable linker, which possesses an Asn-Pro-Val (NPV) sequence sensitive to the human neutrophil elastase (HNE), overexpressed in the tumor microenvironment. This linker permitted the site-specific conjugation of the cell-permeable drug, monomethyl auristatin E (MMAE), onto trastuzumab, using a disulfide re-bridging technology. The resulting ADC was then evaluated in vitro. This conjugate demonstrated retained antigen (Ag) binding affinity and exhibited high subnanomolar potency against Ag-positive tumor cells after internalization, suggesting an intracellular mechanism of linker cleavage. While no internalization and cytotoxic activity of this ADC was observed on Ag-negative cells in classical conditions, the supplementation of exogenous HNE permitted to restore a nanomolar activity on these cells, suggesting an extracellular mechanism of drug release in these conditions. This in vitro proof of concept tends to prove that the NPV sequence could allow a dual intra- and extracellular mechanism of drug release. This work represents a first step in the design of original ADCs with a new dual intra- and extracellular drug delivery system and opens the way to further experimentations to evaluate their full potential in vivo.
Assuntos
Antineoplásicos/química , Imunoconjugados/química , Elastase de Leucócito/metabolismo , Oligopeptídeos/química , Trastuzumab/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Dipeptídeos/química , Dissulfetos/química , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Humanos , Imunoconjugados/farmacologia , Ligação Proteica , Conformação Proteica , Trastuzumab/farmacologiaRESUMO
Antibody-drug conjugates (ADCs) derived from a full immunoglobulin-G (IgG) are associated with suboptimal solid-tumor penetration and Fc-mediated toxicities. Antibody fragment-drug conjugates (FDCs) could be an alternative. Nevertheless, innovative solutions are needed to implant cysteines as conjugation sites in the single-chain fragment variable (scFv) format, which is the backbone from which many other antibody formats are built. In addition, the bioconjugation site has the utmost importance to optimize the safety and efficacy of bioconjugates. Our previous intra-tag cysteine (ITC) strategy consisted of introducing a bioconjugation motif at the C-terminal position of the 4D5.2 scFv, but this motif was subjected to proteolysis when the scFv was produced in CHO cells. Considering these data, using three intra-domain cysteine (IDC) strategies, several parameters were studied to assess the impact of different locations of a site-specific bioconjugation motif in the variable domains of an anti-HER2 scFv. In comparison to the ITC strategy, our new IDC strategy allowed us to identify new fragment-drug conjugates (FDCs) devoid of proteolysis and exhibiting enhanced stability profiles, better affinity, and better ability to kill selectively HER2-positive SK-BR-3 cells in vitro at picomolar concentrations. Thus, this work represents an important optimization step in the design of more complex and effective conjugates.
RESUMO
Antibody-based therapeutics have become a major class of therapeutics with over 120 recombinant antibodies approved or under review in the EU or US. This therapeutic class has experienced a remarkable expansion with an expected acceleration in 2021-2022 due to the extraordinary global response to SARS-CoV2 pandemic and the public disclosure of over a hundred anti-SARS-CoV2 antibodies. Mainly delivered intravenously, alternative delivery routes have emerged to improve antibody therapeutic index and patient comfort. A major hurdle for antibody delivery and efficacy as well as the development of alternative administration routes, is to understand the different natural and pathological barriers that antibodies face as soon as they enter the body up to the moment they bind to their target antigen. In this review, we discuss the well-known and more under-investigated extracellular and cellular barriers faced by antibodies. We also discuss some of the strategies developed in the recent years to overcome these barriers and increase antibody delivery to its site of action. A better understanding of the biological barriers that antibodies have to face will allow the optimization of antibody delivery near its target. This opens the way to the development of improved therapy with less systemic side effects and increased patients' adherence to the treatment.
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Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Anticorpos/uso terapêutico , Humanos , Fatores Imunológicos , Pandemias , RNA ViralRESUMO
We utilized a series of pyrimidine analogues modified at O(2), N-3, and N(4)/O(4) to determine if two B family DNA polymerases, human DNA polymerase α and herpes simplex virus I DNA polymerase, choose whether to polymerize pyrimidine dNTPs using the same mechanisms they use for purine dNTPs. Removing O(2) of a pyrimidine dNTP vastly decreased the level of incorporation by these enzymes and also compromised fidelity in the case of C analogues, while removing O(2) from the templating base had more modest effects. Removing the Watson-Crick hydrogen bonding groups of N-3 and N(4)/O(4) greatly impaired polymerization, both of the resulting dNTP analogues and of natural dNTPs opposite these pyrimidine analogues when present in the template strand. Thus, the Watson-Crick hydrogen bonding groups of a pyrimidine clearly play an important role in enhancing correct dNTP polymerization but are not essential for preventing misincorporation. These studies also indicate that DNA polymerases recognize bases extremely asymmetrically, both in terms of whether they are a purine or pyrimidine and whether they are in the template or are the incoming dNTP. The mechanistic implications of these results with regard to how polymerases discriminate between right and wrong dNTPs are discussed.
Assuntos
DNA Polimerase I/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Proteínas Virais/metabolismo , DNA Polimerase I/química , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , Exodesoxirribonucleases/química , Humanos , Ligação de Hidrogênio , Polimerização , Purinas/química , Pirimidinas/química , Proteínas Virais/químicaRESUMO
Inhibition of protein-DNA interactions represents an attractive strategy to modulate essential cellular functions. We reported the synthesis of unique oligoamide-based foldamers that adopt single helical conformations and mimic the negatively charged phosphate moieties of B-DNA. These mimics alter the activity of DNA interacting enzymes used as targets for cancer treatment, such as DNA topoisomerase I, and they are cytotoxic only in the presence of a transfection agent. The aim of our study was to improve internalization and selective delivery of these highly charged molecules to cancer cells. For this purpose, we synthesized an antibody-drug conjugate (ADC) using a DNA mimic as a payload to specifically target cancer cells overexpressing HER2. We report the bioconjugation of a 16-mer DNA mimic with trastuzumab and its functional validation in breast and ovarian cancer cells expressing various levels of HER2. Binding of the ADC to HER2 increased with the expression of the receptor. The ADC was internalized into cells and was more efficient than trastuzumab at inhibiting their growth in vitro. These results provide proof of concept that it is possible to site-specifically graft high molecular weight payloads such as DNA mimics onto monoclonal antibodies to improve their selective internalization and delivery in cancer cells.
RESUMO
Human DNA primase synthesizes short RNA primers that DNA polymerase alpha then elongates during the initiation of all new DNA strands. Even though primase misincorporates NTPs at a relatively high frequency, this likely does not impact the final DNA product since the RNA primer is replaced with DNA. We used an extensive series of purine and pyrimidine analogues to provide further insights into the mechanism by which primase chooses whether or not to polymerize a NTP. Primase readily polymerized a size-expanded cytosine analogue, 1,3-diaza-2-oxophenothiazine NTP, across from a templating G but not across from A. The enzyme did not efficiently polymerize NTPs incapable of forming two Watson-Crick hydrogen bonds with the templating base with the exception of UTP opposite purine deoxyribonucleoside. Likewise, primase did not generate base pairs between two nucleotides with altered Watson-Crick hydrogen-bonding patterns. Examining the mechanism of NTP polymerization revealed that human primase can misincorporate NTPs via both template misreading and a primer-template slippage mechanism. Together, these data demonstrate that human primase strongly depends on Watson-Crick hydrogen bonds for efficient nucleotide polymerization, much more so than the mechanistically related herpes primase, and provide insights into the potential roles of primer-template stability and base tautomerization during misincorporation.
Assuntos
DNA Primase/química , DNA Primase/metabolismo , Nucleotídeos/química , Pareamento de Bases , Sítios de Ligação , Primers do DNA/química , Primers do DNA/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Dados de Sequência Molecular , Nucleotídeos/metabolismo , Especificidade por SubstratoRESUMO
An armed antibody (antibody-drug conjugate or ADC) is a vectorized chemotherapy, which results from the grafting of a cytotoxic agent onto a monoclonal antibody via a judiciously constructed spacer arm. ADCs have made considerable progress in 10 years. While in 2009 only gemtuzumab ozogamicin (Mylotarg®) was used clinically, in 2020, 9 Food and Drug Administration (FDA)-approved ADCs are available, and more than 80 others are in active clinical studies. This review will focus on FDA-approved and late-stage ADCs, their limitations including their toxicity and associated resistance mechanisms, as well as new emerging strategies to address these issues and attempt to widen their therapeutic window. Finally, we will discuss their combination with conventional chemotherapy or checkpoint inhibitors, and their design for applications beyond oncology, to make ADCs the magic bullet that Paul Ehrlich dreamed of.
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The design of a vehicle frame is largely dependent on the loads applied on the suspension and heavy parts mounting points. These loads can either be estimated through full analytical multibody dynamic simulations, or from semi-analytical simulations in which tire and road sub-models are not included and external vehicle loads, recorded during field testing, are used as inputs to the wheel hubs. Several semi-analytical methods exist, with various modeling architectures, yet, it is unclear how one method over another improves frame loads prediction accuracy. This study shows that a semi-analytical method that constrains the vehicle frame center of gravity movement along a recorded trajectory, using a control algorithm, leads to an accuracy within 1% for predicting frame loads, when compared to reference loads from a full analytical model. The control algorithm computes six degrees of freedom forces and moments applied at the vehicle center of gravity to closely follow the recorded vehicle trajectory. It is also shown that modeling the flexibility of the suspension arms and controlling wheel hub angular velocity both contribute in improving frame loads accuracy, while an acquisition frequency of 200 Hz appears to be sufficient to capture load dynamics for several maneuvers. Knowledge of these loads helps engineers perform appropriate dimensioning of vehicle structural components therefore ensuring their reliability under various driving conditions.
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Antibody-drug conjugates (ADCs) are the spearhead of targeted therapies. According to the technology used, the conjugation of a cytotoxic drug to an antibody can produce suboptimal heterogeneous species, impacting the overall efficacy. Herein, we describe the synthesis of HER2-targeting ADCs with three disulfide rebridging heads, allowing homogeneous and site-specific bioconjugation: dibromomaleimide (DBM), dithiomaleimide (DTM), and hybrid thio-bromomaleimide (TBM) chemical bricks to combine the properties of both previously used heads. The primary purpose of this study was to compare the reactivity of these three chemical bricks in the bioconjugation process. Then, the resulting ADCs were evaluated in terms of physicochemical stability, binding, and biological activity. We have demonstrated that the higher percentage of a drug-to-antibody ratio of 4 was obtained with TBM. Additionally, the reaction time was drastically reduced with TBM in comparison to DTM. The three ADCs showed good binding to HER2 and in vitro cytotoxicity, which validate the TBM structure as an attractive alternative scaffold for rebridging bioconjugation.
RESUMO
Herpes simplex virus-1 primase misincorporates the natural NTPs at frequencies of around one error per 30 NTPs polymerized, making it one of the least accurate polymerases known. We used a series of nucleotide analogues to further test the hypothesis that primase requires Watson-Crick hydrogen bond formation to efficiently polymerize a NTP. Primase could not generate base pairs containing a complete set of hydrogen bonds in an altered arrangement (isoguanine.isocytosine) and did not efficiently polymerize dNTPs completely incapable of forming Watson-Crick hydrogen bonds opposite templating bases incapable of forming Watson-Crick hydrogen bonds. Similarly, primase did not incorporate most NTPs containing hydrophobic bases incapable of Watson-Crick hydrogen bonding opposite natural template bases. However, 2-pyridone NTP and 4-methyl-2-pyridone NTP provided striking exceptions to this rule. The effects of removing single Watson-Crick hydrogen bonding groups from either the NTP or templating bases varied from almost no effect to completely blocking polymerization depending both on the parental base pair (G.C vs A.T/U) and which base pair of the growing primer (second, third, or fourth) was examined. Thus, primase does not absolutely need to form Watson-Crick hydrogen bonds to efficiently polymerize a NTP. Additionally, we found that herpes primase can misincorporate nucleotides both by misreading the template and by a primer-template slippage mechanism. The mechanistic and biological implications of these results are discussed.
Assuntos
Biocatálise , DNA Helicases/metabolismo , DNA Primase/metabolismo , Herpesvirus Humano 1/enzimologia , Proteínas Virais/metabolismo , Pareamento de Bases , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Ligação de Hidrogênio , Estrutura Molecular , Oligodesoxirribonucleotídeos , RNA/biossíntese , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Especificidade por SubstratoRESUMO
An Antibody-Drug Conjugate (armed antibody) is a vectorized chemotherapy that results from the grafting of a cytotoxic agent on a monoclonal antibody thanks to a judiciously designed spacer arm. ADCs have made considerable progress in 10 years. In 2009, only gemtuzumab ozogamicin (Mylotarg®) was used clinically. In 2019, 4 other ADCs have been approved and more than 80 others are in active clinical trials. The first part of this review will focus on Food and Drug Administration-approved Antibody-Drug Conjugates, their limitations as well as their associated toxicity and resistance mechanisms.
TITLE: Les immuno-conjugués en oncologie - Les raisons du succès récent d'une approche ancienne. ABSTRACT: Un anticorps armé (antibody-drug conjugate en anglais) est une chimiothérapie vectorisée qui résulte du greffage d'un agent cytotoxique sur un anticorps monoclonal par l'intermédiaire d'un bras espaceur judicieusement construit. Les anticorps armés ont fait des progrès considérables en 10 ans. En 2009, seul le gemtuzumab ozogamicine (Mylotarg®) était utilisé en clinique. En 2019, 4 autres ADC ont été approuvés par la Food and drug administration et plus de 80 autres sont en études cliniques actives. La première partie de cette revue sera focalisée sur les anticorps armés approuvés, leurs limitations, ainsi que leur toxicité et mécanismes de résistances associés.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Imunoconjugados/uso terapêutico , Oncologia/tendências , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/classificação , Aprovação de Drogas , Gemtuzumab/uso terapêutico , História do Século XX , História do Século XXI , Humanos , Imunoconjugados/classificação , Maitansina/uso terapêutico , Oncologia/história , Oncologia/métodos , Oncologia/normas , Resultado do Tratamento , Estados Unidos , United States Food and Drug AdministrationRESUMO
An Antibody-Drug Conjugate (armed antibody) is a vectorized chemotherapy that results from the grafting of a cytotoxic agent on a monoclonal antibody via a judiciously designed spacer arm. ADCs have made considerable progress in 10 years. In 2009, only gemtuzumab ozogamicin (Mylotarg®) was used clinically. In 2019, 4 other ADCs have been approved and more than 80 others are in active clinical trials. The second part of this review will focus on new emerging strategies to address ADCs drawbacks and attempt to broaden their therapeutic window. Finally, combinations with conventional chemotherapy or checkpoint inhibitors will be discussed, in the pursuit to make Antibody-Drug Conjugates the embodiment of Paul Ehrlich's dream of the magic bullet.
TITLE: Les immunoconjugués en oncologie - Les nouvelles stratégies en développement. ABSTRACT: Un anticorps armé (ADC, antibody-drug conjugate en anglais) est une chimiothérapie vectorisée qui résulte du greffage d'un agent cytotoxique sur un anticorps monoclonal par l'intermédiaire d'un bras espaceur judicieusement construit. Les anticorps armés ont fait des progrès considérables en 10 ans. En 2009, seul le gemtuzumab ozogamicine (Mylotarg®) était utilisé en clinique. En 2019, 4 autres ADC ont été approuvés par la Food and Drug Administration et plus de 80 autres sont en études cliniques actives. La seconde partie de cette revue sera focalisée sur les nouvelles stratégies émergentes pour faire face aux limitations des ADC actuels et pour tenter d'élargir leur fenêtre thérapeutique. Enfin, les combinaisons avec la chimiothérapie classique ou les inhibiteurs de points de contrôles seront discutées, pour tenter de faire des anticorps armés la magic bullet dont rêvait Paul Ehrlich.
Assuntos
Desenvolvimento de Medicamentos/métodos , Imunoconjugados , Oncologia , Neoplasias/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/classificação , Antineoplásicos Imunológicos/isolamento & purificação , Antineoplásicos Imunológicos/uso terapêutico , Desenvolvimento de Medicamentos/tendências , História do Século XXI , Humanos , Imunoconjugados/classificação , Imunoconjugados/isolamento & purificação , Imunoconjugados/uso terapêutico , Oncologia/métodos , Oncologia/tendências , Neoplasias/patologiaRESUMO
Fluorescent labelling of monoclonal antibodies (mAbs) is classically performed by chemical bioconjugation methods. The most frequent labelling technique to generate antibody-fluorophore conjugates (AFCs) involves the bioconjugation onto the mAb lysines of a dye bearing an N-hydroxysuccinimide ester or an isothiocyanate group. However, discrepancies between labelling experiments or kits can be observed, related to reproducibility issues, alteration of antigen binding, or mAb properties. The lack of information on labelling kits and the incomplete characterization of the obtained labelled mAbs largely contribute to these issues. In this work, we generated eight AFCs through either lysine or interchain cysteine cross-linking bioconjugation of green-emitting fluorophores (fluorescein or BODIPY) onto either trastuzumab or rituximab. This strategy allowed us to study the influence of fluorophore solubility, bioconjugation technology, and antibody nature on two known labelling procedures. The structures of these AFCs were thoroughly analyzed by mass spectroscopy, and their antigen binding properties were studied. We then compared these AFCs in vitro by studying their respective spectral properties and stabilities. The shelf stability profiles and sensibility to pH variation of these AFCs prove to be dye-, antibody- and labelling-technology-dependent. Fluorescence emission in AFCs was higher when lysine labelling was used, but cross-linked AFCs were revealed to be more stable. This must be taken into account for the design of any biological study involving antibody labelling.