RESUMEN
Nonspecific ligation methods have been traditionally used to chemically modify immunoglobulins. Site-specific ligation of compounds (toxins or ligands) to antibodies has become increasingly important in the fields of therapeutic antibody-drug conjugates and bispecific antibodies. In this present study, we took advantage of the reported nucleotide-binding pocket (NBP) in the Fab arms of immunoglobulins by developing indole-based, 5-fluoro-2,4-dinitrobenzene-derivatized OBOC peptide libraries for the identification of affinity elements that can be used as site-specific derivatization agents against both mono- and polyclonal antibodies. Ligation can occur at any one of the few lysine residues located at the NBP. Immunoconjugates resulting from such affinity elements can be used as therapeutics against cancer or infectious agents.
Asunto(s)
Inmunoconjugados/química , Inmunoglobulinas/química , Biblioteca de Péptidos , Anticuerpos Biespecíficos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Compuestos Azo/química , Sitios de Unión , Biotina/química , Reactivos de Enlaces Cruzados/química , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulinas/metabolismo , Indoles/química , Nucleótidos/metabolismo , Oligopéptidos/química , Péptidos/química , Péptidos/metabolismo , Trastuzumab/químicaRESUMEN
Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood-brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.
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Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Humanos , Animales , Ratones , Microglía , Barrera Hematoencefálica , Distribución Tisular , Anticuerpos , Encéfalo , Modelos Animales de Enfermedad , Glicoproteínas de Membrana , Receptores Inmunológicos/genéticaRESUMEN
Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TVCD98hc). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATVCD98hc) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATVCD98hc demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATVCD98hc that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TVCD98hc as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.
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Barrera Hematoencefálica , Encéfalo , Animales , Ratones , Distribución Tisular , Anticuerpos , Ingeniería , Macaca fascicularisRESUMEN
In vivo wound healing response post nonablative fractional laser treatment is evaluated. Seven healthy subjects receive treatments with a Fraxel re:store laser system on the forearm with pulse energies ranging from 10 to 70 mJ. The treatment sites are imaged at 1-h increments up to 40 h using confocal microscope z-stacks using 10-mum-depth spacing. At least five individual microscopic treatment zones are imaged per subject, time point, and treatment energy. Images are analyzed for tissue structure and morphology to classify each lesion as healed or not healed, depending on epidermal re-epithelialization at each time point and treatment energy. Probit analysis is used to statistically determine the ED(50) and ED(84) probabilities for a positive dose response (healed lesion) as a function of treatment energy. Confocal observations reveal epidermal keratinocyte migration patterns confirmed with histological analysis using hematoxylin and eosin (HE) and lactate dehydrogenase (LDH) staining at 10 mJ at 0, 7, 16, and 24-h post-treatment. Results indicate that more time is required to conclude re-epithelialization with larger lesion sizes (all less than 500 mum) corresponding to higher treatment energies. For the entire pulse energy range tested, epidermal re-epithelialization concludes between 10 to 22-h post-treatment for ED(50) and 13 to 28 h for ED(84).
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Dermoscopía/métodos , Terapia por Láser/métodos , Microscopía Confocal/métodos , Fenómenos Fisiológicos de la Piel , Piel/citología , Cicatrización de Heridas/fisiología , Humanos , Estadística como AsuntoRESUMEN
Acne vulgaris is a clinically distinct skin condition with evidence suggesting that inflammation plays a critical role in the pathogenesis of this disorder. Treatment of severe inflammatory acne often involves the use of oral antibiotics, sometimes in combination with topical products. Oral antibiotics often result in systemic side effects and the risks of antibiotic resistance, but no commercial topical minocycline is currently available. We have developed a unique, stable, hydrophilic topical gel formulation with fully solubilized minocycline (MNC-H). Minocycline delivered in our hydrophilic gel remained more stable in situ, resulting in less degradation product (4-epiminocycline) than a lipophilic formulation (MNC-L). The hydrophilic nature of our formulation enabled 2-3 fold increase in delivery into the skin ex vivo compared to a lipophilic counterpart, mostly seen in the epidermis and pilosebaceous units. The lipophilic formulation also appeared to be more occlusive, resulting in higher sebum production in minipigs, which may exacerbate acne vulgaris. As our results indicate, a 1, 2% minocycline hydrophilic gel may deliver sufficient drug (>15⯵g/g) to potentially demonstrate clinical efficacy. These findings suggest that topical hydrophilic minocycline gel may provide a novel tool for topical acne therapy.
RESUMEN
The tumor penetration and accumulation of nanoparticle-based drug delivery systems are highly dependent on the particle size. Nanomedicines in the sub-100nm range have been suggested by previous studies to have superior antitumor efficacy on various solid tumors. SN-38 is a very important and highly potent drug for several cancers including colon cancer. However, due to the ultra-flat aromatic structure of SN-38, it is typically very difficult to produce sub-100nm, SN-38-encapsulated nanoparticles without modification of the chemical structure. Here, we report on the successful production of 20-30nm, SN-38-encapsulated photonic micelles for effectively trimodal cancer therapy. Taking advantages of the supramolecular "π-π" stacking and hydrophobicity interaction between SN-38, and a unique class of photonic nanoporphyrin micelles (NPM), the extremely hydrophobic SN-38 was successfully encapsulated into NPM with significantly increased water solubility (up to 500 times). At equivalent dose of drug, photosensitizer and light irradiation, combination therapy with SN-38-encapsulated nanoporphyrin micelles (SN-NPM) enhanced the in vitro antitumor activity by 78 and 350 times over single treatment with SN-38 and phototherapy alone, respectively. Due to the relatively small size, SN-NPM possessed superior long tumor retention time (>5days) and much higher accumulation in tumors than in normal organs, as shown by near-infrared fluorescence (NIRF) imaging. Furthermore, the trimodal therapy (photothermal-, photodynamic- and chemo-therapy) with SN-NPM demonstrated dramatically enhanced in vivo antitumor efficacy over single treatment on nude mice bearing HT-29 colon cancer xenograft. Therefore, these sub-100nm, SN-38-encapsulated photonic micelles show great promise for multimodal cancer therapy.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Camptotecina/análogos & derivados , Neoplasias del Colon/terapia , Nanopartículas , Fototerapia/métodos , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Camptotecina/administración & dosificación , Camptotecina/química , Camptotecina/farmacología , Neoplasias del Colon/patología , Terapia Combinada , Sistemas de Liberación de Medicamentos , Células HT29 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Irinotecán , Masculino , Ratones , Ratones Desnudos , Micelas , Tamaño de la Partícula , Fotones , Fármacos Fotosensibilizantes/administración & dosificación , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The overall prognosis of bladder cancer has not been improved over the last 30 years and therefore, there is a great medical need to develop novel diagnosis and therapy approaches for bladder cancer. We developed a multifunctional nanoporphyrin platform that was coated with a bladder cancer-specific ligand named PLZ4. PLZ4-nanoporphyrin (PNP) integrates photodynamic diagnosis, image-guided photodynamic therapy, photothermal therapy and targeted chemotherapy in a single procedure. PNPs are spherical, relatively small (around 23 nm), and have the ability to preferably emit fluorescence/heat/reactive oxygen species upon illumination with near infrared light. Doxorubicin (DOX) loaded PNPs possess slower drug release and dramatically longer systemic circulation time compared to free DOX. The fluorescence signal of PNPs efficiently and selectively increased in bladder cancer cells but not normal urothelial cells in vitro and in an orthotopic patient derived bladder cancer xenograft (PDX) models, indicating their great potential for photodynamic diagnosis. Photodynamic therapy with PNPs was significantly more potent than 5-aminolevulinic acid, and eliminated orthotopic PDX bladder cancers after intravesical treatment. Image-guided photodynamic and photothermal therapies synergized with targeted chemotherapy of DOX and significantly prolonged overall survival of mice carrying PDXs. In conclusion, this uniquely engineered targeting PNP selectively targeted tumor cells for photodynamic diagnosis, and served as effective triple-modality (photodynamic/photothermal/chemo) therapeutic agents against bladder cancers. This platform can be easily adapted to individualized medicine in a clinical setting and has tremendous potential to improve the management of bladder cancer in the clinic.
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Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Microscopía Fluorescente/métodos , Nanopartículas/administración & dosificación , Fotoquimioterapia/métodos , Porfirinas/administración & dosificación , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Animales , Línea Celular Tumoral , Terapia Combinada/métodos , Doxorrubicina/administración & dosificación , Humanos , Ratones , Ratones Endogámicos C57BL , Terapia Molecular Dirigida/métodos , Nanopartículas/química , Péptidos Cíclicos/administración & dosificación , Fármacos Fotosensibilizantes/administración & dosificación , Fototerapia/métodos , Nanomedicina Teranóstica/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: α3ß1 integrin is overexpressed in several types of human cancer and is associated with poor prognosis, metastasis, and resistance to cancer treatment. We previously identified a cyclic peptide ligand LXY1 that specifically binds to the α3ß1 integrin on human glioblastoma U-87MG cells. Here, we optimized LXY1 through one-bead one-compound combinatorial library screening and site-specific modifications to improve its in vivo binding property. METHODS: Three bead libraries were synthesized and whole-cell binding assays were performed. The binding capacity of individual peptide ligands against different tumor cells was determined by flow cytometry and confirmed by optical imaging. A complex joining biotinylated ligand with streptavidin-Cy5.5 was used for in vivo target imaging in both subcutaneous and orthotopic U-87MG xenograft mouse models. RESULTS: LXY30, a cyclic peptide with the sequence cdG-Phe(3,5-diF)-G-Hyp-NcR, emerged as the most potent and selective ligand for the α3 subunit of α3ß1 integrin with improved in vitro and in vivo tumor-targeting effects compared to LXY1 in U-87MG cells. LXY30 is considerably stable in plasma as demonstrated in an in vitro stability study in 90 % human plasma. LXY30 also binds to several other known α3ß1 integrin-expressing glioblastoma, lung, and breast cancer cell lines with various affinities. CONCLUSIONS: Our data support further investigating the role of LXY30 as a human tumor-targeting peptide ligand for systemic and intracranial delivery of imaging agents and cancer therapeutics.
RESUMEN
Multifunctional nanoparticles with combined diagnostic and therapeutic functions show great promise towards personalized nanomedicine. However, attaining consistently high performance of these functions in vivo in one single nanoconstruct remains extremely challenging. Here we demonstrate the use of one single polymer to develop a smart 'all-in-one' nanoporphyrin platform that conveniently integrates a broad range of clinically relevant functions. Nanoporphyrins can be used as amplifiable multimodality nanoprobes for near-infrared fluorescence imaging (NIRFI), magnetic resonance imaging (MRI), positron emission tomography (PET) and dual modal PET-MRI. Nanoporphyrins greatly increase the imaging sensitivity for tumour detection through background suppression in blood, as well as preferential accumulation and signal amplification in tumours. Nanoporphyrins also function as multiphase nanotransducers that can efficiently convert light to heat inside tumours for photothermal therapy (PTT), and light to singlet oxygen for photodynamic therapy (PDT). Furthermore, nanoporphyrins act as programmable releasing nanocarriers for targeted delivery of drugs or therapeutic radio-metals into tumours.
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Portadores de Fármacos/química , Nanoestructuras/química , Nanoestructuras/uso terapéutico , Porfirinas/química , Nanomedicina Teranóstica/métodos , Animales , Línea Celular Tumoral/efectos de los fármacos , Dendrímeros/química , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Portadores de Fármacos/farmacocinética , Femenino , Fluorescencia , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Ratones Desnudos , Ratones Transgénicos , Nanoestructuras/administración & dosificación , Fotoquimioterapia/métodos , Porfirinas/sangre , Porfirinas/farmacocinética , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Espectroscopía Infrarroja Corta/métodos , Nanomedicina Teranóstica/instrumentaciónRESUMEN
Mammalian homeobox gene Emx family is involved in the development of the rostral brain. Loss-of-function studies suggest that, despite the agenesis of corpus callosum, the Emx1 mutants display relatively modest defects compared to the Emx2 mutants. However, the role of the Emx1 in neurogenesis and brain function has never been explored. We used unbiased stereology to determine the number of proliferating progenitors and immature neurons in the adult neurogenic zones. Although previous studies have established that the formation of the dentate gyrus (DG) requires Emx2, we found that the adult Emx1 mutants also exhibited a smaller DG, reduced number of proliferating progenitor cells and immature neurons in the DG, in contrast to the indistinguishable level of neurogenesis in the subventricular zone when compared to the wild type mice. In view of the involvement of callosal projection neurons in mediating interhemispheric crosstalk and spatial coupling between the limbs, and the importance of DG in hippocampus-dependent function in learning and memory, we assessed motor and cognitive functions. Emx1 deletion impaired performance on a forelimb skill reaching task and attenuated training induced hippocampal neurogenesis, but it did not affect motor activity or basic motor function as evaluated in the open field, wire hanging and rotor rod tests. Unexpectedly, the adult Emx1 mutant mice did not exhibit impairment in spatial learning and memory in the Barnes maze test. Our data suggest that deletion of the Emx1 gene reduces hippocampal neurogenesis and affects higher motor function that requires extensive learning.