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The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.
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COVID-19 , Humanos , Ligantes , COVID-19/metabolismo , Ceramidas/metabolismo , Pulmão/metabolismo , Endotélio Vascular/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Transporte/metabolismo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
A major challenge of targeted molecular imaging and drug delivery in cancer is establishing a functional combination of ligand-directed cargo with a triggered release system. Here we develop a hydrogel-based nanotechnology platform that integrates tumor targeting, photon-to-heat conversion, and triggered drug delivery within a single nanostructure to enable multimodal imaging and controlled release of therapeutic cargo. In proof-of-concept experiments, we show a broad range of ligand peptide-based applications with phage particles, heat-sensitive liposomes, or mesoporous silica nanoparticles that self-assemble into a hydrogel for tumor-targeted drug delivery. Because nanoparticles pack densely within the nanocarrier, their surface plasmon resonance shifts to near-infrared, thereby enabling a laser-mediated photothermal mechanism of cargo release. We demonstrate both noninvasive imaging and targeted drug delivery in preclinical mouse models of breast and prostate cancer. Finally, we applied mathematical modeling to predict and confirm tumor targeting and drug delivery. These results are meaningful steps toward the design and initial translation of an enabling nanotechnology platform with potential for broad clinical applications.
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Antineoplásicos/administração & dosagem , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Modelos Animais de Doenças , Imagem Multimodal , Nanotecnologia , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/tratamento farmacológico , Animais , Sistemas de Liberação de Medicamentos , Feminino , Raios Infravermelhos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ressonância de Plasmônio de SuperfícieRESUMO
Inflammatory breast carcinoma (IBC) is one of the most lethal forms of human breast cancer, and effective treatment for IBC is an unmet clinical need in contemporary oncology. Tumor-targeted theranostic approaches are emerging in precision medicine, but only a few specific biomarkers are available. Here we report up-regulation of the 78-kDa glucose-regulated protein (GRP78) in two independent discovery and validation sets of specimens derived from IBC patients, suggesting translational promise for clinical applications. We show that a GRP78-binding motif displayed on either bacteriophage or adeno-associated virus/phage (AAVP) particles or loop-grafted onto a human antibody fragment specifically targets orthotopic IBC and other aggressive breast cancer models in vivo. To evaluate the theranostic value, we used GRP78-targeting AAVP particles to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) transgene, obtaining simultaneous in vivo diagnosis through PET imaging and tumor treatment by selective activation of the prodrug ganciclovir at tumor sites. Translation of this AAVP system is expected simultaneously to image, monitor, and treat the IBC phenotype and possibly other aggressive (e.g., invasive and/or metastatic) subtypes of breast cancer, based on the inducible cell-surface expression of the stress-response chaperone GRP78, and possibily other cell-surface receptors in human tumors.
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Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC.
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We performed combinatorial peptide library screening in vivo on a novel human prostate cancer xenograft that is androgen-independent and induces a robust osteoblastic reaction in bonelike matrix and soft tissue. We found two peptides, PKRGFQD and SNTRVAP, which were enriched in the tumors, targeted the cell surface of androgen-independent prostate cancer cells in vitro, and homed to androgen receptor-null prostate cancer in vivo. Purification of tumor homogenates by affinity chromatography on these peptides and subsequent mass spectrometry revealed a receptor for the peptide PKRGFQD, α-2-macroglobulin, and for SNTRVAP, 78-kDa glucose-regulated protein (GRP78). These results indicate that GRP78 and α-2-macroglobulin are highly active in osteoblastic, androgen-independent prostate cancer in vivo. These previously unidentified ligand-receptor systems should be considered for targeted drug development against human metastatic androgen-independent prostate cancer.
Assuntos
Neoplasias Ósseas/secundário , Osteogênese , Peptídeos/química , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia de Afinidade , Progressão da Doença , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Ligantes , Masculino , Camundongos , Camundongos SCID , Nanotecnologia , Transplante de Neoplasias , Neoplasias de Próstata Resistentes à Castração/patologia , Ligação Proteica , Proteômica , Receptores Androgênicos/metabolismo , alfa-Macroglobulinas/metabolismoRESUMO
Development of therapy against infections caused by antibiotic-resistant pathogens is a major unmet need in contemporary medicine. In previous work, our group chemically modified an antimicrobial peptidomimetic motif for targeted applications against cancer and obesity. Here, we show that the modified motif per se is resistant to proteolytic degradation and is a candidate antiinfective agent. We also show that the susceptibility of microorganisms to the drug is independent of bacterial growth phase. Moreover, this peptidomimetic selectively interferes with the integrity and function of the microbial surface lipid bilayer, data indicative that bacterial death results from membrane disruption followed by dissipation of membrane potential. Finally, we demonstrate two potential translational applications: use against biofilms and synergy with antibiotics in use. In summary, we introduce the mechanism of action and the initial evaluation of a prototype drug and a platform for the development of D-enantiomer antimicrobial peptidomimetics that target bacterial membranes of certain gram-negative problem pathogens with promising translational applications.
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Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Membrana Celular/metabolismo , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Sequência de Aminoácidos , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Bactérias/ultraestrutura , Biofilmes/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Cinética , Bicamadas Lipídicas/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Estereoisomerismo , Fatores de TempoRESUMO
BACKGROUND: Receptors in tumor blood vessels are attractive targets for ligand-directed drug discovery and development. The authors have worked systematically to map human endothelial receptors ("vascular zip codes") within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand-binding motif to the interleukin-11 receptor alpha (IL-11Rα) in the human vasculature. METHODS: The authors generated a ligand-directed, peptidomimetic drug (bone metastasis-targeting peptidomimetic-11 [BMTP-11]) for IL-11Rα-based human tumor vascular targeting. Preclinical studies (efficacy/toxicity) included evaluating BMTP-11 in prostate cancer xenograft models, drug localization, targeted apoptotic effects, pharmacokinetic/pharmacodynamic analyses, and dose-range determination, including formal (good laboratory practice) toxicity across rodent and nonhuman primate species. The initial BMTP-11 clinical development also is reported based on a single-institution, open-label, first-in-class, first-in-man trial (National Clinical Trials number NCT00872157) in patients with metastatic, castrate-resistant prostate cancer. RESULTS: BMTP-11 was preclinically promising and, thus, was chosen for clinical development in patients. Limited numbers of patients who had castrate-resistant prostate cancer with osteoblastic bone metastases were enrolled into a phase 0 trial with biology-driven endpoints. The authors demonstrated biopsy-verified localization of BMTP-11 to tumors in the bone marrow and drug-induced apoptosis in all patients. Moreover, the maximum tolerated dose was identified on a weekly schedule (20-30 mg/m(2) ). Finally, a renal dose-limiting toxicity was determined, namely, dose-dependent, reversible nephrotoxicity with proteinuria and casts involving increased serum creatinine. CONCLUSIONS: These biologic endpoints establish BMTP-11 as a targeted drug candidate in metastatic, castrate-resistant prostate cancer. Within a larger discovery context, the current findings indicate that functional tumor vascular ligand-receptor targeting systems may be identified through direct combinatorial selection of peptide libraries in cancer patients.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/prevenção & controle , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Peptídeos/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Neoplasias Ósseas/secundário , Esquema de Medicação , Humanos , Subunidade alfa de Receptor de Interleucina-11/efeitos dos fármacos , Rim/efeitos dos fármacos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Peptídeos/farmacologia , Proteinúria/induzido quimicamente , Resultado do TratamentoRESUMO
Dendrimers have several featured advantages over other nanomaterials as drug carriers, such as well-defined structure, specific low-nanometer size, and abundant peripheral derivable groups, etc. However, these advantages have not been fully exploited yet to optimize their biological performance, especially tumor penetration, which is a shortcoming of current nanomaterials. Here we show the syntheses of a new class of oligo(ethylene glycol) (OEG)-based thermosensitive dendrimers up to the fourth generation. Each dendrimer shows monodisperse structure. OEG/poly(ethylene glycol) (PEG) moieties with different precise lengths were introduced to the periphery of the fourth-generation dendrimer followed by an antitumor agent, gemcitabine (GEM). The biodistributions of the GEM-conjugated dendrimers were investigated by micro positron emission tomography and multispectral optoacoustic tomography imaging techniques and compared with that of GEM-conjugated poly(amidoamine) (PAMAM). The GEM-conjugated dendrimer with the longest peripheral PEG segments exhibited the most desirable tumor accumulation and penetration and thus had significantly higher antitumor activity than the GEM-conjugated PAMAM.
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Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dendrímeros/síntese química , Desoxicitidina/análogos & derivados , Portadores de Fármacos/síntese química , Polietilenoglicóis/síntese química , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Dendrímeros/química , Dendrímeros/farmacologia , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Feminino , Citometria de Fluxo , Camundongos , Microscopia Eletrônica de Transmissão , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Tomografia por Emissão de Pósitrons , GencitabinaRESUMO
Brain research depends strongly on imaging for assessing function and disease in vivo. We examine herein multispectral opto-acoustic tomography (MSOT), a novel technology for high-resolution molecular imaging deep inside tissues. MSOT illuminates tissue with light pulses at multiple wavelengths and detects the acoustic waves generated by the thermoelastic expansion of the environment surrounding absorbing molecules. Using spectral unmixing analysis of the data collected, MSOT can then differentiate the spectral signatures of oxygenated and deoxygenated hemoglobin and of photo-absorbing agents and quantify their concentration. By being able to detect absorbing molecules up to centimeters deep in the tissue it represents an ideal modality for small animal brain imaging, simultaneously providing anatomical, hemodynamic, functional, and molecular information. In this work we examine the capacity of MSOT in cross-sectional brain imaging of mice. We find unprecedented optical imaging performance in cross-sectional visualization of anatomical and physiological parameters of the mouse brain. For example, the potential of MSOT to characterize ischemic brain areas was demonstrated through the use of a carbon dioxide challenge. In addition, indocyanine green (ICG) was injected intravenously, and the kinetics of uptake and clearance in the vasculature of the brain was visualized in real-time. We further found that multiparameter, multispectral imaging of the growth of U87 tumor cells injected into the brain could be visualized through the intact mouse head, for example through visualization of deoxygenated hemoglobin in the growing tumor. We also demonstrate how MSOT offers several compelling features for brain research and allows time-dependent detection and quantification of brain parameters that are not available using other imaging methods without invasive procedures.
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Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Imagem Molecular/métodos , Técnicas Fotoacústicas/métodos , Tomografia/métodos , Animais , Modelos Animais de Doenças , Feminino , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos NusRESUMO
OBJECTIVE: To evaluate the long-term patency rate of the arteriovenous angioaccess (AVA) with interposition of either autologous or prosthetic material as a last option for vascular access in the upper extremity. METHODS: This is a retrospective chart review study of all patients who received an AVA with autologous saphenous vein (SV Group, n = 38) or prosthetic material (PTFE Group, n = 25) as a conduit from the year 1996 to 2020 in the Radboud University Medical Center (Radboudumc). Data were retrospectively extracted from two prospectively updated local databases for vascular access, one for haemodialysis (HD) and one for parenteral nutrition (PN). When required, the medical records of each patient were used. Data were eventually collected anonymously and analysed in SPSS 25. Kaplan-Meier life-tables were used for the statistical analysis. RESULTS: Primary patency at 12 and 48 months was 30% and 20% in the SV group and 45% and 14% in the PTFE group. No significant difference was shown in the median primary patency rate (p = 0.715). Secondary patency at 12 and 48 months was 63% and 39% in the SV group and 55% and 19% in the PTFE group. This was considered a significant difference in median secondary patency in favour of the SV with 41.16 ± 17.67 months against 13.77 ± 10.22 months for PTFE (p = 0.032). The incidence of infection was significantly lower in the SV group (p = 0.0002). A Kaplan-Meier curve could not detect a significant difference in secondary patency between the access for haemodialysis and the access for parenteral nutrition. The secondary patency of the SV in parenteral nutrition access, was significantly higher when compared with PTFE (p = 0.004). CONCLUSION: The SV can be preferred over PTFE when conduit material is needed for long-term vascular access for HD or PN treatment due to its higher secondary patency and lower infection risk.
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Implante de Prótese Vascular , Humanos , Implante de Prótese Vascular/efeitos adversos , Estudos Retrospectivos , Veia Safena , Grau de Desobstrução Vascular , Diálise Renal/efeitos adversos , Politetrafluoretileno , Prótese Vascular/efeitos adversos , Resultado do Tratamento , Oclusão de Enxerto Vascular/etiologiaRESUMO
This white paper summarizes the recommendations of the absorption, distribution, metabolism, and excretion (ADME) Subcommittee of the Oligonucleotide Safety Working Group for the characterization of absorption, distribution, metabolism, and excretion of oligonucleotide (ON) therapeutics in nonclinical studies. In general, the recommended approach is similar to that for small molecule drugs. However, some differences in timing and/or scope may be warranted due to the greater consistency of results across ON classes as compared with the diversity among small molecule classes. For some types of studies, a platform-based approach may be appropriate; once sufficient data are available for the platform, presentation of these data should be sufficient to support development of additional ONs of the same platform. These recommendations can serve as a starting point for nonclinical study design and foundation for discussions with regulatory agencies.
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Oligonucleotídeos , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos/farmacocinéticaRESUMO
Many in vitro and in vivo models are used in pharmacological research to evaluate the role of targeted proteins in a disease. Understanding the translational relevance and limitation of these models for analyzing a drug's disposition, pharmacokinetic/pharmacodynamic (PK/PD) profile, mechanism, and efficacy, is essential when selecting the most appropriate model of the disease of interest and predicting clinically efficacious doses of the investigational drug. Selected animal models used in ophthalmology, infectious diseases, oncology, autoimmune diseases, and neuroscience are reviewed here. Each area has specific challenges around translatability and determination of an efficacious dose: new patient-specific dosing methods may help overcome these limitations.
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Drogas em Investigação , Oncologia , Animais , Modelos BiológicosRESUMO
PURPOSE: To develop an efficient and safe strategy to introduce a therapeutic gene into target cells in vivo for cancer therapy. The overall efficiency is based on proper selection of the delivery vector and expressed protein. METHODS: A plasmid coding for a specific cytotoxic fusion peptide, p14ARF-TAT, was evaluated in a xenograft mouse tumor model. The expressed peptide consisted of three domains, a secretory signal, a membrane permeability segment and a cytotoxic fragment. Gene expression was verified in U87-MG cells by Western blot and cytotoxicity confirmed with CyQuant assay. To improve the delivery, a FGF2 targeting peptide, MQLPLATC, was incorporated into the vector, which was evaluated using a luciferase-expressing plasmid. RESULTS: The luciferase activity in vitro was two-fold higher with the targeted formulations, and cytotoxicity was three-fold higher with expression of the p14ARF-TAT protein. A murine xenograph model of human glioma (U87-MG cells) tumors was used to address in vivo activity. FGF2-targeted lipoplexes demonstrated increased tumor volume reduction as compared to non-targeted formulations. RT-PCR and Western blot of tumor homogenizes indicated p14ARF-TAT expression in tumors along with other tissues. CONCLUSION: p14ARF-TAT was cytotoxic and is a promising approach when combined with an efficient targeting.
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Fator 2 de Crescimento de Fibroblastos/genética , Produtos do Gene tat/genética , Técnicas de Transferência de Genes , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Proteínas Recombinantes de Fusão/genética , Proteína Supressora de Tumor p14ARF/genética , Animais , Apoptose , Western Blotting , Cátions , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular , Marcação de Genes , Terapia Genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Marcação In Situ das Extremidades Cortadas , Luciferases/genética , Camundongos , Camundongos Nus , Estrutura Molecular , Transplante de Neoplasias , Tamanho da Partícula , Plasmídeos/administração & dosagem , Plasmídeos/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , TransfecçãoRESUMO
Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.
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New challenges and other topics in non-clinical safety testing of biotherapeutics were presented and discussed at the nineth European BioSafe Annual General Membership meeting in November 2019. The session topics were selected by European BioSafe organization committee members based on recent company achievements, agency interactions and new data obtained in the non-clinical safety testing of biotherapeutics, for which data sharing would be of interest and considered as valuable information. The presented session topics ranged from strategies of in vitro testing, immunogenicity prediction, bioimaging, and developmental and reproductive toxicology (DART) assessments to first-in-human (FIH) dose prediction and bioanalytical challenges, reflecting the entire space of different areas of expertise and different molecular modalities. During the 9th meeting of the European BioSafe members, the following topics were presented and discussed in 6 main sessions (with 3 or 4 presentations per session) and in three small group breakout sessions: 1) DART assessment with biotherapeutics: what did we learn and where to go?; 2) Non-animal testing strategies; 3) Seeing is believing: new frontiers in imaging; 4) Predicting immunogenicity during early drug development: hope or despair?; 5) Challenges in FIH dose projections; and 6) Non-canonical biologics formats: challenges in bioanalytics, PKPD and biotransformation for complex biologics formats. Small group breakout sessions were organized for team discussion about 3 specific topics: 1) Testing of cellular immune function in vitro and in vivo; 2) MABEL approach (toxicology and pharmacokinetic perspective); and 3) mRNA treatments. This workshop report presents the sessions and discussions at the meeting.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , HumanosRESUMO
Triple-negative breast cancer (TNBC) is an aggressive tumor with limited treatment options and poor prognosis. We applied the in vivo phage display technology to isolate peptides homing to the immunosuppressive cellular microenvironment of TNBC as a strategy for non-malignant target discovery. We identified a cyclic peptide (CSSTRESAC) that specifically binds to a vitamin D receptor, protein disulfide-isomerase A3 (PDIA3) expressed on the cell surface of tumor-associated macrophages (TAM), and targets breast cancer in syngeneic TNBC, non-TNBC xenograft, and transgenic mouse models. Systemic administration of CSSTRESAC to TNBC-bearing mice shifted the cytokine profile toward an antitumor immune response and delayed tumor growth. Moreover, CSSTRESAC enabled ligand-directed theranostic delivery to tumors and a mathematical model confirmed our experimental findings. Finally, in silico analysis showed PDIA3-expressing TAM in TNBC patients. This work uncovers a functional interplay between a cell surface vitamin D receptor in TAM and antitumor immune response that could be therapeutically exploited.
Assuntos
Antineoplásicos/farmacologia , Oligopeptídeos/farmacologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Macrófagos Associados a Tumor/efeitos dos fármacos , Proteína de Ligação a Vitamina D/metabolismo , Animais , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Biológicos , Isomerases de Dissulfetos de Proteínas/genética , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Proteína de Ligação a Vitamina D/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A peptide analog, 4-fluorobenzoyl-RR-(L-3-(2-naphthyl)alanine)-CYEK-(L-citrulline)-PYR-(L-citrulline)-CR, covalently linked to a phospholipid, was used for targeting a lipid-based gene delivery vehicle to CXCR4(+)-cells. Characterization of transfection activity was done in vitro using a transformed rat glioma cell line (RG2) that expresses CXCR4. The substitution of the targeting lipid at increasing mole percentages in the place of helper lipids yielded a progressive increase in reporter gene expression, reaching a maximum of 2.5 times the control value at 20 mol% of ligand. The substitution of helper lipids with cysteine-derivatized phospholipid analog or phosphatidylethanolamine resulted in a progressive decrease in transfection activity, with complete inactivation of the complex occurring at 20 mol%. A DNA dose-response with 10 mol% of lipopeptide reduced the effective DNA dose at least fivefold with regard to the number of transfected cells and >20-fold with regard to the amount of gene expression. Gene transfer to rat endothelial cells was studied in the context of an arterial organ culture. Mesenteric arteries were cannulated and maintained in culture for up to 4 days. CXCR4 cell-surface expression on endothelial cells was induced after overnight incubation with vascular endothelial growth factor (VEGF). Gene transfer studies showed that only the peptide-targeted lipoplexes transfected the endothelium, and only after CXCR4 had been induced with VEGF. These results demonstrate that non-viral transfection complexes can be targeted to cells expressing CXCR4, and that gene transfer is dependent upon cell surface receptor expression levels.
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Células Endoteliais/metabolismo , Peptídeos/química , Receptores CXCR4/genética , Transfecção/métodos , Animais , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Citrulina/análogos & derivados , Citrulina/química , Células Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/química , Vetores Genéticos/genética , Glioma/genética , Glioma/patologia , Naftalenos/química , Técnicas de Cultura de Órgãos , Fosfolipídeos/química , Ratos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8+ T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.
Assuntos
Anticorpos Biespecíficos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Anticorpos Biespecíficos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Proliferação de Células/fisiologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Humanos , Imunoterapia , Linfonodos/imunologia , Linfonodos/metabolismo , Neoplasias/imunologia , Neoplasias/terapiaRESUMO
Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 µM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.
RESUMO
There is a need for contrast agents for non-invasive diagnostic imaging of tumors. Herein, Multispectral Optoacoustic Tomography (MSOT) was employed to evaluate phthalocyanines commonly used in photodynamic therapy as photoacoustic contrast agents. We studied the photoacoustic activity of three water-soluble phthalocyanine photosensitizers: phthalocyanine tetrasulfonic acid (PcS4), Zn(II) phthalocyanine tetrasulfonic acid (ZnPcS4) and Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4) in phantom and in tumor-bearing mice to investigate the biodistribution and fate of the phthalocyanines in the biological tissues. PcS4 was observed to grant good contrast between the different reticuloendothelial organs and accumulate in the tumor within an hour of post-administration. ZnPcS4 and AlPcS4 offered little contrast in photoacoustic signals between the organs. PcS4 is a promising photoacoustic contrast agent and can be exploited as a photodiagnostic agent.