Littérature scientifique sur le sujet « Macromolecular recombinant drug »

Transdermal drug administration presents several advantages and it is therefore favorable as an alternative drug delivery route. However, transdermal delivery of biopharmaceutical drugs is made difficult by the skin barrier. Microneedle application and iontophoresis are strategies which can be used to overcome this barrier. Therefore, recombinant human growth hormone (rhGH) was used as a model macromolecular drug and was transdermally delivered using microneedle application and iontophoresis. Methylene blue staining, stereomicroscopy and scanning electron microscope (SEM) imaging were used to characterize the microchannels produced. To optimize the iontophoresis protocol, the effects of molecular charge and current density on transdermal delivery were evaluated in an in vitro permeation study using excised rat skin tissues. Using the optimized iontophoresis protocol, the combination effects of iontophoretic delivery via microchannels were evaluated in three different experimental designs. The flux obtained with anodal iontophoresis in citrate buffer was approximately 10-fold higher that that with cathodal iontophoresis in phosphate buffered saline (PBS). Flux also increased with current density in anodal iontophoresis. The combination of iontophoresis and microneedle application produced higher flux than single application. These results suggest that anodal iontophoresis with higher current density enhances the permeation of macromolecules through microchannels created by microneedles. In conclusion, the combination of iontophoresis and microneedles is a potential strategy for the enhancement of transdermal delivery of macromolecular drugs.

Filoviruses are virulent pathogens that cause deadly haemorrhagic fever in humans and non-human primates. There is currently no approved drug or vaccine to tackle this disease. Two vaccine platforms that use adenovirus vectors have completed phase I studies, while a recombinant vesicular stomatitis virus-based vaccine has successfully complet-ed a phase III trial. Intricate macromolecular therapeutics have also been developed, most notably those based on anti-bodies or interfering RNA or RNA-surrogates. Most small molecules active against filoviruses have not yet advanced to clinical trials, except favipiravir, which was proven to be safe, and GS-5734, which has entered trials.

1. Introduction 292. Landmarks in NMR of macromolecules 322.1 Protein structures and methods development 322.1.1 Sequential assignment method 322.1.2 Triple-resonance experiments 342.1.3 Structures of large proteins 362.2 Protein–nucleic acid complexes 372.3 RNA structures 382.4 Membrane-bound systems 393. NMR spectroscopy today 403.1 State-of-the-art structure determination 413.2 New methods 443.2.1 Residual dipolar couplings 443.2.2 Direct detection of hydrogen bonds 443.2.3 Spin labeling 453.2.4 Segmental labeling 463.3 Protein complexes 473.4 Mobility studies 503.5 Determination of time-dependent structures 523.6 Drug discovery 534. The future of NMR 544.1 The ease of structure determination 544.2 The ease of making recombinant protein 554.3 Post-translationally modified proteins 554.4 Approaches to large and/or membrane-bound proteins 564.5 NMR in structural genomics 564.6 Synergy of NMR and crystallography in protein structure determination 565. Conclusion 576. Acknowledgements 577. References 57Since the publication of the first complete solution structure of a protein in 1985 (Williamson et al. 1985), tremendous technological advances have brought nuclear magnetic resonance spectroscopy to the forefront of structural biology. Innovations in magnet design, electronics, pulse sequences, data analysis, and computational methods have combined to make NMR an extremely powerful technique for studying biological macromolecules at atomic resolution (Clore & Gronenborn, 1998). Most recently, new labeling and pulse techniques have been developed that push the fundamental line-width limit for resolution in NMR spectroscopy, making it possible to obtain high-field spectra with better resolution than ever before (Dötsch & Wagner, 1998). These methods are facilitating the study of systems of ever-increasing complexity and molecular weight.

Enabling challenging applications of nanomedicine and precision medicine in the treatment of neurodegenerative disorders requires deeper investigations of nanocarrier-mediated biomolecular delivery for neuronal targeting and recovery. The successful use of macromolecular biotherapeutics (recombinant growth factors, antibodies, enzymes, synthetic peptides, cell-penetrating peptide–drug conjugates, and RNAi sequences) in clinical developments for neuronal regeneration should benefit from the recent strategies for enhancement of their bioavailability. We highlight the advances in the development of nanoscale materials for drug delivery in neurodegenerative disorders. The emphasis is placed on nanoformulations for the delivery of brain-derived neurotrophic factor (BDNF) using different types of lipidic nanocarriers (liposomes, liquid crystalline or solid lipid nanoparticles) and polymer-based scaffolds, nanofibers and hydrogels. Self-assembled soft-matter nanoscale materials show favorable neuroprotective characteristics, safety, and efficacy profiles in drug delivery to the central and peripheral nervous systems. The advances summarized here indicate that neuroprotective biomolecule-loaded nanoparticles and injectable hydrogels can improve neuronal survival and reduce tissue injury. Certain recently reported neuronal dysfunctions in long-COVID-19 survivors represent early manifestations of neurodegenerative pathologies. Therefore, BDNF delivery systems may also help in prospective studies on recovery from long-term COVID-19 neurological complications and be considered as promising systems for personalized treatment of neuronal dysfunctions and prevention or retarding of neurodegenerative disorders.

Abstract Single molecule drug delivery systems have failed to yield functional therapeutic outcomes, triggering investigations into multi-molecular drug delivery vehicles. In the context of skin fibrosis, although multi-drug systems have been assessed, no system has assessed molecular combinations that directly and specifically reduce cell proliferation, collagen synthesis and transforming growth factor β1 (TGFβ1) expression. Herein, a core–shell collagen type I hydrogel system was developed for the dual delivery of a TGFβ trap, a soluble recombinant protein that inhibits TGFβ signalling, and Trichostatin A (TSA), a small molecule inhibitor of histone deacetylases. The antifibrotic potential of the dual delivery system was assessed in an in vitro skin fibrosis model induced by macromolecular crowding (MMC) and TGFβ1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance liquid chromatography analyses revealed that ∼50% of the TGFβ trap and ∼30% of the TSA were released from the core and shell compartments, respectively, of the hydrogel system after 10 d (longest time point assessed) in culture. As a direct consequence of this slow release, the core (TGFβ trap)/shell (TSA) hydrogel system induced significantly (p < 0.05) lower than the control group (MMC and TGFβ1) collagen type I deposition (assessed via SDS-PAGE and immunocytochemistry), α smooth muscle actin (αSMA) expression (assessed via immunocytochemistry) and cellular proliferation (assessed via DNA quantification) and viability (assessed via calcein AM and ethidium homodimer-I staining) after 10 d in culture. On the other hand, direct TSA-TGFβ supplementation induced the lowest (p < 0.05) collagen type I deposition, αSMA expression and cellular proliferation and viability after 10 d in culture. Our results illustrate the potential of core–shell collagen hydrogel systems for sustained delivery of antifibrotic molecules.

The Shigella invasin complex or Invaplex vaccine is a unique subunit approach to generate a protective immune response. Invaplex is a large, macromolecular complex consisting of the major Shigella antigens: lipopolysaccharide (LPS) and the invasion plasmid antigen (Ipa) proteins B and C. Over the past several decades, the vaccine has progressed from initial observations through pre-clinical studies to cGMP manufacture and clinical evaluations. The Invaplex product maintains unique biological properties associated with the invasiveness of virulent shigellae and also presents both serotype-specific epitopes, as well as highly conserved invasin protein epitopes, to the immunized host. The vaccine product has evolved from a native product isolated from wild-type shigellae (native Invaplex) to a more defined vaccine produced from purified LPS and recombinant IpaB and IpaC (artificial Invaplex). Each successive “generation” of the vaccine is derived from earlier versions, resulting in improved immunogenicity, homogeneity and effectiveness. The current vaccine, detoxified artificial Invaplex (InvaplexAR-Detox), was developed for parenteral administration by incorporating LPS with under-acylated lipid A. InvaplexAR-Detox has demonstrated an excellent safety and immunogenicity profile in initial clinical studies and is advancing toward evaluations in the target populations of children and travelers to endemic countries.

Hematological malignancies, also referred to as blood cancers, are a group of diseases involving abnormal cell growth and persisting in the blood, lymph nodes, or bone marrow. The development of new targeted therapies including small molecule inhibitors, monoclonal antibodies, bispecific T cell engagers, antibody-drug conjugates, recombinant immunotoxins, and, finally, Chimeric Antigen Receptor T (CAR-T) cells has improved the clinical outcomes for blood cancers. In this review, we summarized 52 drugs that were divided into small molecule and macromolecule agents, approved by the Food and Drug Administration (FDA) in the period between 2011 and 2021 for the treatment of hematological malignancies. Forty of them have also been approved by the European Medicines Agency (EMA). We analyzed the FDA-approved drugs by investigating both their structures and mechanisms of action. It should be emphasized that the number of targeted drugs was significantly higher (46 drugs) than chemotherapy agents (6 drugs). We highlight recent advances in the design of drugs that are used to treat hematological malignancies, which make them more effective and less toxic.

A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.

Abstract Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilia patients with inhibitors. However, rFVIIa is cleared from the circulation relatively fast, with circulating half-life of about 2–4 h, requiring repeated administration of rFVIIa for the effective treatment. Therefore, development of FVIIa analogs that could remain in the circulation for a longer period of time would be of a great value for improving the treatment options of rFVIIa. e.g., by prophylaxis. PEGylation of plasma proteins was shown to extend their circulatory half-lives but the PEGylation may also disrupt macromolecular interactions. In the present study we characterized the interaction of two glycoPEGylated analogs of rFVIIa, rFVIIa-10K PEG and rFVIIa-40K PEG, with its cofactor tissue factor (TF), substrate factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). Both the PEGylated FVIIa analogs exhibited similar amidolytic activity as of wild-type rFVIIa (wt-rFVIIa) in the absence or presence of relipidated TF. The analogs were as effective as wt-rFVIIa in activating FX in the absence of TF. No significant differences were found between the PEGylated rFVIIa analogs and wt-rFVIIa in TF-dependent FX activation at saturating concentrations of rFVIIa, however, at lower concentrations of rFVIIa (10 to 50 pM), rFVIIa-10K PEG and rFVIIa-40K PEG activated FX at a slightly lower rate, 50% and 75%, respectively, of wt-rFVIIa. Further studies revealed that both AT/heparin and TFPI inhibited the PEGylated rFVIIa-TF complexes effectively but slightly at a lower rate compared to that was noted for wt-rFVIIa-TF. TFPI-Xa inhibited the PEGylated rFVIIa-TF and wt-rFVIIa-TF at a similar rate. On unperturbed HUVEC, wt-FVIIa (10 nM) could activate FX, albeit slowly, (1.7 nM/h) and the PEGylated rFVIIa activated FX even at much lower rates (0.23 nM/h for rFVIIa-10K PEG and 0.15 nM/h for rFVIIa-40K PEG). On stimulated HUVEC expressing TF, the PEGylated rFVIIa variants were slightly less effective at lower concentrations compared to wt-rFVIIa in activating FX, but no significant differences were found among them in activating factor X at saturating concentrations of rFVIIa (80–100 nM/h). The PEGylated rFVIIa analogs bound to cell surface TF were inhibited by TFPI-Xa complex at a similar rate as that was observed for wt-rFVIIa (IC50 in nM: 0.102 ± 0.032 for wt-rFVIIa, 0.111 ± 0.024 for rFVIIa-10K PEG, and 0.096 ± 0.019 for rFVIIa-40K PEG). AT/heparin inhibited rFVIIa-10K PEG bound to endothelial cell TF at a similar rate as it inhibited wt-rFVIIa (IC50 in μg/ml: wt-rFVIIa, 3.42 ± 068; rFVIIa-10K PEG, 3.56 ± 0.073), but the inhibition rate was slightly lower for rFVIIa-40K PEG bound to TF (IC50 5.92 ± 0.44 μg/ml). Overall, our present data suggest that long-acting PEGylated FVIIa analogs retain full enzymatic activity and can interact TF and FX effectively, and are inhibited by AT/heparin and TFPI-Xa as for wt-rFVIIa. Although the pegylated rFVIIa variants exhibited somewhat lower affinity towards TF, this may not critically affect the TF-driven FXa generation. Further work is needed to fully characterize these molecules.

L’impiego di agenti citotossici capaci di riconoscere in modo specifico molecole presenti sulla superficie di una cellula tumorale rappresenta una tecnica molto promettente per il trattamento di diverse neoplasie ed è particolarmente efficace per la cura di leucemie e linfomi. Appartengono a questa classe di sostanze le immunotossine, proteine composte da un dominio di derivazione anticorpale e da un dominio tossico solitamente di origine batterica o vegetale: l’anticorpo lega selettivamente l’antigene bersaglio e, a seguito di endocitosi, consente il rilascio all’interno della cellula della componente tossica che interferisce con i processi metabolici necessari alla sopravvivenza cellulare. Tra i diversi marcatori di superficie associati a leucemie e linfomi, uno dei target molecolari di maggior interesse per questo tipo di approccio terapeutico, è il CD22, una glicoproteina di membrana con funzioni co-recettoriali la cui espressione è limitata ai linfociti B. Il presente lavoro di tesi descrive la generazione e la successiva caratterizzazione funzionale di una immunotossina in cui il motivo di legame alla superficie cellulare è costituito da un frammento anticorpale a singola catena (scFv) specifico per l’antigene CD22, mentre l’azione citotossica è svolta da una forma tronca dell’esotossina A di Pseudomonas aeruginosa, proteina in grado di inibire il meccanismo di sintesi proteica eucariotica. Attraverso il clonaggio delle sequenze codificanti per le regioni variabili della catena pesante (VH) e della catena leggera (VL) di un anticorpo monoclonale murino anti-CD22, è stato possibile assemblare il costrutto che codifica per l’scFv ed esprimere la proteina ricombinante in un sistema di espressione eterologo quale Escherichia coli. Mediante analisi di immunofluorescenza e saggi immunoenzimatici è stato verificato che il frammento anticorpale possiede una buona affinità di legame all’antigene e mantiene le stesse caratteristiche di specificità mostrate dall’anticorpo monoclonale parentale. Successivamente l’scFv è stato unito attraverso fusione genetica al dominio enzimatico della tossina batterica. L’immunotossina risultante, espressa in Escherichia coli, è stata estratta a partire da aggregati insolubili accumulati nel compartimento citosolico del batterio e purificata tramite cromatografia di affinità con rese di circa 1-2 mg per litro di coltura batterica. La proteina ricombinante possiede proprietà di legame specifiche per l’antigene non dissimili da quelle osservate per l’scFv, inoltre, secondo quanto accertato mediante saggi di citotossicità, induce in modo selettivo l’inibizione della proliferazione di linee cellulari esprimenti la proteina CD22 con una IC50 di 1- 10 nM (per IC50 si intende la concentrazione in grado di inbire il 50% della proliferazione IV cellulare). Essa sembra quindi conservare inalterate sia le caratteristiche di riconoscimento specifico del frammento anticorpale che l’attività enzimatica della tossina. Ulteriori studi serviranno a valutare l’opportunità di sottoporre l’immunotossina ad un processo di ottimizzazione finalizzato all’ottenimento di un nuovo farmaco biotecnologico per il trattamento di neoplasie ematologiche nell’uomo.
The use of cytotoxic agents capable to selectively target surface molecules on a malignant cell is a promising approach for the treatment of cancer, especially hematologic malignacies. Immunotoxins, in particular, are polypeptides comprising an antibody-derived domain and a toxic portion, usually represented by a bacterial or plant toxin: the antibody specifically binds a target antigen and, following endocytosis, delivers the toxic payload to the interior of the cell, interfering with fundamental metabolic pathways. Among several leukemia/lymphoma-associated surface antigens, one of the most attractive molecular targets for this kind of therapeutic strategy is CD22, a membrane glycoprotein with coreceptor functions, whose expression is restricted to B lymphocytes. The present thesis describes the construction and characterization of a recombinant immunotoxin in which the binding domain is represented by a CD22-specific single-chain antibody fragment (scFv), while the cytotoxic activity is carried out by a truncated version of Pseudomonas aeruginosa Exotoxin A, a bacterial toxin that inhibits the mechanism of protein synthesis in eukaryotes. Through the molecular cloning of sequences coding for the variable domains of heavy and light chains (VH and VL, respectively) of an anti-CD22 murine monoclonal antibody, it was possible to assemble a plasmid construct coding for a scFv that was eventually produced in a bacterial expression system. Immunofluorecence analysis on CD22-positive cells and immunoenzymatic assays on the purified antigen proved that the antibody fragment maintains the binding specificity of the parental monoclonal antibody, exhibiting a fairly good affinity for CD22. The scFv was later genetically fused to the enzymatic domain of a bacterial toxin. The resulting immunotoxin was expressed in Escherichia coli and recovered from insoluble cytoplasmic aggregates after purification by affinity chromatography, with yields of 1-2 mg from a 1 litre culture. The binding properties of the recombinant immunotoxin are comparable to those of the scFv; V moreover, as ascertained through cell-proliferation assays, it can selectively poison CD22- expressing cells with an IC50 (i.e. concentration inhibiting 50% of the maximal cell proliferation) around 1-10 nM. It can be concluded that our anti-CD22 immunotoxin combines the binding qualities of the scFv antibody and the potent enzymatic activity of the bacterial toxin. After further characterization we will explore the opportunity to start a process of molecular optimization, aiming at the construction of a novel biotechnological drug for the treatment of hematological malignancies in humans.

DNA-FACE™ (DNA Fragment Amplification & Concatemeric Expressed Nucleic Acids and Proteins) is a universal biotechnological platform, developed as Escherichia coli (E. coli) system. It is based on the ordered, head-to-tail directional ligation of the amplified DNA fragments. The technology enables the construction of targeted biomolecules - genetically programmed, concatemeric DNA, RNA, and proteins, designed to fit a particular task. The constructed, “artificial” (never seen in Nature) tandem repeat macromolecules, with specialized functions, may contain up to 500 copies of monomeric units. The technology greatly exceeds the current capabilities of chemical gene synthesis. The vector-enzymatic DNA fragment amplification assembles the DNA segments, forming continuous Open Reading Frames (ORFs). The obtained ORFs are ready for high-level expression in E. coli without a need for subcloning. The presented method has potential applications in pharmaceutical industry and tissue engineering, including vaccines, biological drugs, drug delivery systems, mass-production of peptide-derived biomaterials, industrial and environmental processes. The technology has been patented worldwide and used successfully in the construction of anti-HBV vaccines, pro-regenerative biological drugs and, recently, the anti-SARS-CoV-2 vaccine. The anti-SARS-CoV-2 vaccine, developed using the DNA-FACE™ technology, is nontoxic and induces strong immunological response to recombinant human spike and nucleocapsid proteins, as shown in animal studies.