RESUMEN
Antibody-drug conjugates, nanoparticles, and liposomes have been used for anticancer drug delivery. The success of targeted killing of cancer cells relies heavily on the selectivity of the drug delivery systems. In most systems, antibodies or their fragments were used as targeting ligands. In this study, we have investigated the potential for protein-based octomeric chemically self-assembled nanorings (CSANs) to be used for anticancer drug delivery. The CSANs are composed of a DHFR-DHFR fusion protein incorporating an EGFR-targeting fibronectin and the anticancer drug MMAE conjugated through a C-terminal farnesyl azide. The anti-EGFR-MMAE CSANs were shown to undergo rapid internalization and have potent cytotoxicity to cancer cells across a 9000-fold difference in EGFR expression. In addition, anti-EGFR-MMAE CSANs were shown to induce immunological cell death. Thus, multivalent and modular CSANs are a potential alternative anticancer drug delivery platform with the capability of targeting tumor cells with heterogeneous antigen expression while activating the anticancer immune response.
Asunto(s)
Antineoplásicos , Sistemas de Liberación de Medicamentos , Muerte Celular Inmunogénica , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Receptores ErbB/metabolismo , Receptores ErbB/inmunología , Muerte Celular Inmunogénica/efectos de los fármacos , Nanopartículas/química , Nanoestructuras/químicaRESUMEN
Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymesâprotein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)âthat catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.
Asunto(s)
Química Clic , Farnesiltransferasa , Norbornanos , Prenilación de Proteína , Norbornanos/química , Farnesiltransferasa/metabolismo , Humanos , AnimalesRESUMEN
The design of imaging agents with a high fluorine content is necessary for overcoming the challenges of low sensitivity in 19F magnetic resonance imaging (MRI)-based molecular imaging. Chemically self-assembled nanorings (CSANs) provide a strategy to increase the fluorine content through multivalent display. We previously reported an 19F NMR-based imaging tracer, in which case a CSAN-compatible epidermal growth factor receptor (EGFR)-targeting protein E1-dimeric dihydrofolate (E1-DD) was bioconjugated to a highly fluorinated peptide. Despite good 19F NMR performance in aqueous solutions, a limited signal was observed in cell-based 19F NMR using this monomeric construct, motivating further design. Here, we design several new E1-DD proteins bioconjugated to peptides of different fluorine contents. Flow cytometry analysis was used to assess the effect of variable fluorinated peptide sequences on the cellular binding characteristics. Structure-optimized protein, RTC-3, displayed an optimal spectral performance with high affinity and specificity for EGFR-overexpressing cells. To further improve the fluorine content, we next engineered monomeric RTC-3 into CSAN, η-RTC-3. With an approximate eightfold increase in the fluorine content, multivalent η-RTC-3 maintained high cellular specificity and optimal 19F NMR spectral behavior. Importantly, the first cell-based 19F NMR spectra of η-RTC-3 were obtained bound to EGFR-expressing A431 cells, showing a significant amplification in the signal. This new design illustrated the potential of multivalent fluorinated CSANs for future 19F MRI molecular imaging applications.
Asunto(s)
Flúor , Imagen por Resonancia Magnética , Flúor/química , Espectroscopía de Resonancia Magnética , Proteínas , Péptidos , Receptores ErbB/metabolismoRESUMEN
The design of imaging agents with high fluorine content is essential for overcoming the challenges associated with signal detection limits in 19F MRI-based molecular imaging. In addition to perfluorocarbon and fluorinated polymers, fluorinated peptides offer an additional strategy for creating sequence-defined 19F magnetic resonance imaging (MRI) imaging agents with a high fluorine signal. Our previously reported unstructured trifluoroacetyllysine-based peptides possessed good physiochemical properties and could be imaged at high magnetic field strength. However, the low detection limit motivated further improvements in the fluorine content of the peptides as well as removal of nonspecific cellular interactions. This research characterizes several new highly fluorinated synthetic peptides composed of highly fluorinated amino acids. 19F NMR analysis of peptides TB-1 and TB-9 led to highly overlapping, intense fluorine resonances and acceptable aqueous solubility. Flow cytometry analysis and fluorescence microscopy further showed nonspecific binding could be removed in the case of TB-9. As a preliminary experiment toward developing molecular imaging agents, a fluorinated EGFR-targeting peptide (KKKFFKK-ßA-YHWYGYTPENVI) and an EGFR-targeting protein complex E1-DD bioconjugated to TB-9 were prepared. Both bioconjugates maintained good 19F NMR performance in aqueous solution. While the E1-DD-based imaging agent will require further engineering, the success of cell-based 19F NMR of the EGFR-targeting peptide in A431 cells supports the potential use of fluorinated peptides for molecular imaging.
Asunto(s)
Flúor , Imagen por Resonancia Magnética , Flúor/química , Espectroscopía de Resonancia Magnética , Péptidos , Receptores ErbBRESUMEN
The homeostasis of cellular activities is essential for the normal functioning of living organisms. Hence, the ability to regulate the fates of cells is of great significance for both fundamental chemical biology studies and therapeutic development. Despite the notable success of small-molecule drugs that normally act on cellular protein functions, current clinical challenges have highlighted the use of macromolecules to tune cell function for improved therapeutic outcomes. As a class of hybrid biomacromolecules gaining rapidly increasing attention, protein conjugates have exhibited great potential as versatile tools to manipulate cell function for therapeutic applications, including cancer treatment, tissue engineering, and regenerative medicine. Therefore, recent progress in the design and assembly of protein conjugates used to regulate cell function is discussed in this review. The protein conjugates covered here are classified into three different categories based on their mechanisms of action and relevant applications: (1) regulation of intercellular interactions; (2) intervention in intracellular biological pathways; (3) termination of cell proliferation. Within each genre, a variety of protein conjugate scaffolds are discussed, which contain a diverse array of grafted molecules, such as lipids, oligonucleotides, synthetic polymers, and small molecules, with an emphasis on their conjugation methodologies and potential biomedical applications. While the current generation of protein conjugates is focused largely on delivery, the next generation is expected to address issues of site-specific conjugation, in vivo stability, controllability, target selectivity, and biocompatibility.
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Polímeros , Proteínas , Proteínas/química , Polímeros/química , Sustancias Macromoleculares , Oligonucleótidos , LípidosRESUMEN
Inspired by the natural intercellular material-transfer process of trans-endocytosis or trogocytosis, we proposed that targeted farnesylated chemically self-assembled nanorings (f-CSANs) could serve as a biomimetic trogocytosis vehicle for engineering directional cargo transfer between cells, thus allowing cell-cell interactions to be monitored and facilitating cell-cell communications. The membranes of sender cells were stably modified by hydrophobic insertion with the targeted f-CSANs, which were efficiently transferred to receiver cells expressing the appropriate receptors by endocytosis. CSAN-assisted cell-cell cargo transfer (C4T) was demonstrated to be receptor specific and dependent on direct cell-cell interactions, the rate of receptor internalization, and the level of receptor expression. In addition, C4T was shown to facilitate cell-to-cell delivery of an apoptosis inducing drug, as wells as antisense oligonucleotides. Taken together, the C4T approach is a potentially versatile biomimetic trogocytosis platform that can be deployed as a macro-chemical biological tool for monitoring cell-cell interactions and engineering cell-cell communications.
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Nanoestructuras , Nanoestructuras/química , Comunicación Celular , Biomimética , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
Protein-based conjugates have been extensively utilized in various biotechnological and therapeutic applications. In order to prepare homogeneous conjugates, site-specific modification methods and efficient purification strategies are both critical factors to be considered. The development of general and facile conjugation and purification strategies is therefore highly desirable. Here, we apply a capture and release strategy to create protein conjugates based on Designed Ankyrin Repeat Proteins (DARPins), which are engineered antigen-binding proteins with prominent affinity and selectivity. In this case, DARPins that target the epithelial cell adhesion molecule (EpCAM), a diagnostic cell surface marker for many types of cancer, were employed. The DARPins were first genetically modified with a C-terminal CVIA sequence to install an enzyme recognition site and then labeled with an aldehyde functional group employing protein farnesyltransferase. Using a capture and release strategy, conjugation of the labeled DARPins to a TAMRA fluorophore was achieved with either purified proteins or directly from crude E. coli lysate and used in subsequent flow cytometry and confocal imaging analysis. DARPin-MMAE conjugates were also prepared yielding a construct manifesting an IC50 of 1.3 nM for cell killing of EpCAM positive MCF-7 cells. The method described here is broadly applicable to enable the streamlined one-step preparation of protein-based conjugates.
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Repetición de Anquirina , Proteínas de Repetición de Anquirina Diseñadas , Aldehídos/metabolismo , Transferasas Alquil y Aril , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas/químicaRESUMEN
Few therapeutic options have been made available for treating central nervous system tumors, especially upon recurrence. Recurrent medulloblastoma is uniformly lethal with no approved therapies. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3. However, due to several therapy-related toxicities and reports of tumor escape, the full potential of targeting this pan-cancer antigen has yet to be realized. Here, we designed and characterized bispecific chemically self-assembling nanorings (CSANs) that target the T cell receptor, CD3ε, and tumor associated antigen, B7-H3, derived from the humanized 8H9 single chain variable fragment. We show that the αB7-H3-αCD3 CSANs increase T cell infiltration and facilitate selective cytotoxicity of B7-H3+ medulloblastoma spheroids and that activity is independent of target cell MHC class I expression. Importantly, nonspecific T cell activation against the ONS 2303 medulloblastoma cell line can be reduced by tuning the valency of the αCD3 targeted monomer in the oligomerized CSAN. Intraperitoneal injections of αB7-H3-αCD3 bispecific CSANs were found to effectively cross the blood-tumor barrier into the brain and elicit significant antitumor T cell activity intracranially as well as systemically in an orthotopic medulloblastoma model. Moreover, following treatment with αB7-H3-αCD3 CSANs, intratumoral T cells were found to primarily have a central memory phenotype that displayed significant levels of characteristic activation markers. Collectively, these results demonstrate the ability of our multivalent, bispecific CSANs to direct potent antitumor T cell responses and indicate its potential utility as an alternative or complementary therapy for immune cell targeting of B7-H3+ brain tumors.
Asunto(s)
Neoplasias Encefálicas , Neoplasias Cerebelosas , Meduloblastoma , Humanos , Linfocitos T , Meduloblastoma/tratamiento farmacológico , Activación de Linfocitos , Antígenos de Neoplasias , Línea Celular TumoralRESUMEN
Overexpression of the epidermal growth factor receptor (EGFR) on various cancers makes it an important target for cancer immunotherapy. We recently demonstrated that single-chain variable fragment-based bispecific chemically self-assembled nanorings (CSANs) can successfully modify T cell surfaces and function as prosthetic antigen receptors (PARs) allowing selective targeting of tumor antigens while incorporating a dissociation mechanism of the rings. Here, we report the generation of anti-EGFR fibronectin (FN3)-based PARs with high yield, rapid protein production, predicted low immunogenicity, and increased protein stability. We demonstrated the cytotoxicity of FN3-PARs successfully while evaluating FN3 affinities, CSAN valencies, and antigen expression levels. Using an orthotopic breast cancer model, we showed that FN3-PARs can suppress tumor growth with no adverse effects and FN3-PARs reduced immunosuppressive programmed cell death ligand-1 (PD-L1) expression by downregulating EGFR signaling. These results demonstrate the potential of FN3-PARs to direct selective T cell-targeted tumor killing and to enhance antitumor T cell efficacy by modulating the tumor microenvironment.
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Anticuerpos Biespecíficos/uso terapéutico , Fibronectinas/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias/terapia , Anticuerpos de Cadena Única/uso terapéutico , Linfocitos T/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Antígeno B7-H1/antagonistas & inhibidores , Complejo CD3/inmunología , Línea Celular Tumoral , Regulación hacia Abajo , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Femenino , Fibronectinas/inmunología , Humanos , Inhibidores de Puntos de Control Inmunológico/inmunología , Ratones Endogámicos NOD , Ratones SCID , Receptor de Muerte Celular Programada 1/metabolismo , Transducción de Señal/efectos de los fármacos , Anticuerpos de Cadena Única/inmunologíaRESUMEN
Multicellular biology is dependent on the control of cell-cell interactions. These concepts have begun to be exploited for engineering of cell-based therapies. Herein, we detail the use of a multivalent lipidated scaffold for the rapid and reversible manipulation of cell-cell interactions. Chemically self-assembled nanorings (CSANs) are formed via the oligomerization of bivalent dihydrofolate reductase (DHFR2) fusion proteins using a chemical dimerizer, bis-methotrexate. With targeting proteins fused onto the DHFR2 monomers, the CSANs can target specific cellular antigens. Here, anti-EGFR or anti-EpCAM fibronectin-DHFR2 monomers incorporating a CAAX-box sequence were enzymatically prenylated, then assembled into the corresponding CSANs. Both farnesylated and geranylgeranylated CSANs efficiently modified the cell surface of lymphocytes and remained bound to the cell surface with a half-life of >3 days. Co-localization studies revealed a preference for the prenylated nanorings to associate with lipid rafts. The presence of antigen targeting elements in these bifunctional constructs enabled them to specifically interact with target cells while treatment with trimethoprim resulted in rapid CSAN disassembly and termination of the cell-cell interactions. Hence, we were able to determine that activated PBMCs modified with the prenylated CSANs caused irreversible selective cytotoxicity toward EGFR-expressing cells within 2 hours without direct engagement of CD3. The ability to disassemble these nanostructures in a temporally controlled manner provides a unique platform for studying cell-cell interactions and T cell-mediated cytotoxicity. Overall, antigen-targeted prenylated CSANs provide a general approach for the regulation of specific cell-cell interactions and will be valuable for a plethora of fundamental and therapeutic applications.
RESUMEN
Nanosuspensions of drugs are nanosized colloidal dispersions of pure particles. In contrast to conventional nanoparticles, the particles in nanosuspensions feature 100% drug loading. Stiripentol (STP) is an effective drug for severe myoclonic epilepsy of infancy (SMEI); however, because of its low water solubility, high oral doses of STP, up to 50 mg per kg per day in two or three divided doses, must be administered to patients, compromising therapy outcomes. Here, we report STP nanosuspensions (STP-Ns) stabilized with denatured soybean protein isolate (SPI) as a stabilizer to promote the absorption of STP and thus improve therapeutic outcomes. STP-Ns with a drug loading of up to 50% (w/w) and a diameter of 150 nm were successfully prepared. Importantly, in the presence of denatured SPI as a stabilizer, the drug state in the nanosuspensions was tunable by drug loading: low drug loading resulted in the formation of amorphous drug nanoparticles while high drug loading greater than 3.22% (w/w) in formulation induced the formation of nanosuspensions with the coexistence of amorphous and crystalline drug. This new nanosuspension formulation was related to the fact that the protein-drug complex exhibited a much stronger affinity for the drug particles over the protein itself. Interestingly, via the transcytosis pathway, the STP-Ns penetrated across the intestinal barrier into the systemic circulation, with the duodenum as the predominant absorption site. The bioavailability of the STP-Ns was 4-fold as great as that of raw crystals. The discovery of this mechanism for the use of globular protein as a stabilizer for nanosuspensions provides a new route for the preparation of amorphous drug nanoparticles. This work offers a new strategy to widen the application of globular protein and nanosuspensions of insoluble active compounds in drug delivery.
RESUMEN
Oral drug delivery is the most preferred route for patients; however, the low solubility of drugs and the resultant poor absorption compromise the benefits of oral administration. On the other hand, for years, the overwhelmingly accepted mechanism for enhanced oral absorption using lipid nanocarriers was based on the process of lipid digestion and drug solubilization in the small intestine. Few reports indicated that other bypass pathways are involved in drug absorption in the gastrointestinal tract (GIT) for oral delivery of nanocarriers. Herein, we report a new nanoemulsion system with a denatured globular protein with a diameter of 30 nm, soybean protein isolates (SPI), and bile salt as emulsifiers, aiming to enhance the absorption of insoluble drugs and explore other pathways for absorption. A BCS class II drug, fenofibrate (FB), was used as the model drug. The SPI and bile salt-coated Ns with a diameter of approximately 150 nm were prepared via a high-pressure homogenizing procedure. Interestingly, the present Ns could be converted to solid dosage form using fluid-bed coating technology, maintaining a nanoscale size. Most importantly, in a model of in situ rat intestinal perfusion, Ns could penetrate across the intestinal epithelial barrier into the systemic circulation and then obtain biodistribution into other tissues. In addition, Ns significantly improved FB oral absorption, exhibited as a greater than 2- and 2.5-fold increase in Cmax and AUC0-t, respectively, compared to the suspension formulation. Overall, the present Ns are promising nanocarriers for the oral delivery of insoluble drugs, and the penetration of intact Ns across the GIT barrier into systemic circulation may be a new strategy for improved drug absorption with the use of nanocarriers.
Asunto(s)
Ácidos y Sales Biliares/administración & dosificación , Ácidos y Sales Biliares/química , Portadores de Fármacos/química , Fenofibrato/sangre , Fenofibrato/farmacocinética , Absorción Intestinal , Nanopartículas/administración & dosificación , Proteínas de Soja/química , Administración Oral , Animales , Ácidos y Sales Biliares/farmacocinética , Disponibilidad Biológica , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/farmacocinética , Emulsiones/administración & dosificación , Emulsiones/efectos adversos , Emulsiones/química , Emulsiones/farmacocinética , Fenofibrato/administración & dosificación , Fenofibrato/química , Humanos , Nanopartículas/química , Nanopartículas/ultraestructura , Tamaño de la Partícula , Desnaturalización Proteica , Ratas , Solubilidad , Proteínas de Soja/administración & dosificación , Proteínas de Soja/farmacocinética , Suspensiones/farmacocinética , Distribución Tisular , Agua/químicaRESUMEN
A quick wireless label-free detection of disease-related C-reactive proteins (CRPs) using a 200-microm-long microelectromechanical systems (MEMS) microcantilever housed in a 7 x 7 mm(2) reaction chamber with a safe reusable feature is reported. The assay time ranges from about 30 min to 3 h, depending on accuracy. The deflection of the microcantilever due to specific CRP-antiCRP binding is detected using a position-sensitive detector. The converted bio-signal is transmitted by a custom designed wireless amplitude-shift-keying (ASK) transceiver IC fabricated in a 0.18 microm CMOS process. CRP concentrations from 1 microg/mL to 500 microg/mL can be detected. A 0.2-Hz 1-V ac signal instead of traditional bases/acids is applied to the bio-MEMS sensor to unbind the CRP from the microcantilever for reusability.