RESUMEN
Photodynamic therapy (PDT) and immunotherapy have been often adopted for ovarian cancer therapy, yet their application is limited by the high recurrence rate and toxic side effects. Intriguingly, nanoparticles contribute to enhancing the performance of PDT. Here, we investigated the synthesis of HER-2-Nanobody (Nb)-conjugated human serum albumin (HSA) incorporated with chlorin (Ce6) and catalase (CAT) (Nb@HCC), and analyzed the synergic effect of Nb@HCC-mediated PDT and immunotherapy for SK-OV-3 tumors. The Ce6 and CAT were incorporated into HSA to construct the HCC nanoparticles. HER-2-Nanobody was the purified bacterial crude extract, and conjugated with HCC to prepare Nb@HCC via heterodisulfide. The effects of Nb@HCC with near infrared ray (NIR) irradiation on moderating hypoxia and hypoxia inducible factor-1α(HIF-1α) expression were evaluated in the SK-OV-3 cells and tumor tissues. A SK-OV-3 tumor-bearing model was developed, where the synergistic effect of Nb@HCC-mediated PDT and anti-CTLA-4 therapy was investigated. Nb@HCC with a 660 nm laser irradiation could induce massive reactive oxygen species and trigger apoptosis in SK-OV-3 cells. Nb@HCC and PDT promoted danger-associated molecular patterns (DAMPs), which indicated immunogenic cell death and maturation of dendritic cells in the SK-OV-3 cells. Irradiated by NIR, Nb@HCC alleviated the hypoxia and decreased the expression of HIF-1α. The Nb@HCC-mediated PDT and anti-CTLA-4 therapy synergically inhibited the progression of distant tumor, and induced T cell infiltration. Biosafety tests suggested that Nb@HCC would not cause damage to the major organs with less toxicity and side effects. To conclude, a combination of Nb@HCC-mediated PDT and anti-CTLA-4 therapy could inhibit the progression of distant tumor to attain remarkable therapeutic outcomes.
Asunto(s)
Hipoxia de la Célula , Neoplasias Ováricas/metabolismo , Fotoquimioterapia/métodos , Anticuerpos de Dominio Único , Animales , Antineoplásicos/química , Antineoplásicos/toxicidad , Catalasa , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/efectos de la radiación , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Porfirinas , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/toxicidadRESUMEN
Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Inmunotoxinas/uso terapéutico , Terapia Molecular Dirigida , Neoplasias de la Próstata/tratamiento farmacológico , Anticuerpos de Dominio Único/uso terapéutico , Animales , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos de Superficie/inmunología , Antineoplásicos Inmunológicos/toxicidad , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Glutamato Carboxipeptidasa II/inmunología , Humanos , Inmunotoxinas/toxicidad , Masculino , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes de Fusión/toxicidad , Anticuerpos de Dominio Único/toxicidad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The safety of 2 single domain antibodies (dAbs) was evaluated by inhalation toxicology studies in the cynomolgus monkey. In the first case study, a 14-day repeat-dose study evaluating an anti-thymic stromal lymphopoietin (anti-TSLP) dAb resulted in minimal mononuclear inflammatory cell infiltrates in the lungs, increases in lymphocytes in bronchoalveolar lavage fluid, and development of antidrug antibodies (ADAs). In a 6-week inhalation study, there was an increase in incidence and/or severity of mononuclear cell infiltrates in the lung, increased cellularity in the tracheobronchial lymph node (TBLN), and development of ADA. The second case study evaluated a change in duration of inhalation dosing, a different route of exposure (intravenous or IV), and recovery following an off-dose period with an anti-tumor necrosis factor receptor 1 dAb. A 7-day repeat-dose inhalation study and a 14-day IV study produced no microscopic effects in the lung, whereas a 14-day inhalation study resulted in moderate increases in pulmonary perivascular/peribronchiolar/alveolar lymphocytic infiltrates and increased cellularity in the TBLN, with partial and full recovery, respectively, after 14 days. The lung and lymph node findings seen after inhalation of either dAb were considered secondary to the immunogenic response to a human protein and were considered nonadverse.
Asunto(s)
Anticuerpos de Dominio Único , Administración por Inhalación , Animales , Líquido del Lavado Bronquioalveolar , Pulmón , Macaca fascicularis , Anticuerpos de Dominio Único/toxicidadRESUMEN
BACKGROUND: CD47, the integrin-related protein, plays an important role in immune resistance and escape of tumor cells. Antibodies blocking the CD47/SIRPα signal pathway can effectively stimulate macrophage-mediated phagocytosis of tumor cells, which becomes a promising approach for tumor immunotherapy. Nanobodies (Nbs) derived from camelid animals are emerging as a new force in antibody therapy. RESULTS: HuNb1-IgG4, an innovative anti-CD47 nanobody, was developed with high affinity and specificity. It effectively enhanced macrophage-mediated phagocytosis of tumor cells in vitro and showed potent anti-ovarian and anti-lymphoma activity in vivo. Importantly, HuNb1-IgG4 did not induce the agglutination of human red blood cells (RBCs) in vitro and exhibited high safety for hematopoietic system in cynomolgus monkey. In addition, HuNb1-IgG4 could be produced on a large scale in CHO-S cells with high activity and good stability. Also, we established anti-CD47/CD20 bispecific antibody (BsAb) consisted of HuNb1 and Rituximab, showing more preference binding to tumor cells and more potent anti-lymphoma activity compared to HuNb1-IgG4. CONCLUSIONS: Both of HuNb1-IgG4 and anti-CD47/CD20 BsAb are potent antagonists of CD47/SIRPα pathway and promising candidates for clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Antígeno CD47/inmunología , Anticuerpos de Dominio Único/farmacología , Anticuerpos de Dominio Único/toxicidad , Animales , Línea Celular , Técnicas de Visualización de Superficie Celular , Femenino , Humanos , Inmunoglobulina G/metabolismo , Macaca fascicularis , Ratones Endogámicos NOD , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Lung-targeting drugs are thought to be potential therapies of refractory lung diseases by maximizing local drug concentrations in the lung to avoid systemic circulation. However, a major limitation in developing lung-targeted drugs is the acquirement of lung-specific ligands. Pulmonary surfactant protein A (SPA) is predominantly synthesized by type II alveolar epithelial cells, and may serve as a potential lung-targeting ligand. Here, we generated recombinant rat pulmonary SPA (rSPA) as an antigen and immunized an alpaca to produce two nanobodies (the smallest naturally occurring antibodies) specific for rSPA, designated Nb6 and Nb17. To assess these nanobodies' potential for lung targeting, we evaluated their specificity to lung tissue and toxicity in mice. Using immunohistochemistry, we demonstrated that these anti-rSPA nanobodies selectively bound to rat lungs with high affinity. Furthermore, we intravenously injected fluorescein isothiocyanate-Nb17 in nude mice and observed its preferential accumulation in the lung to other tissues, suggesting high affinity of the nanobody for the lung. Studying acute and chronic toxicity of Nb17 revealed its safety in rats without causing apparent histological alterations. Collectively, we have generated and characterized lung-specific nanobodies, which may be applicable for lung drug delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , Proteína A Asociada a Surfactante Pulmonar/inmunología , Anticuerpos de Dominio Único , Animales , Ratones , Ratones Desnudos , Ratas , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/toxicidadRESUMEN
UNLABELLED: Nanobodies are the smallest fully functional antigen-binding antibody fragments possessing ideal properties as probes for molecular imaging. In this study we labeled the anti-human epidermal growth factor receptor type 2 (HER2) Nanobody with (68)Ga via a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivative and assessed its use for HER2 iPET imaging. METHODS: The 2Rs15dHis6 Nanobody and the lead optimized current-good-manufacturing-practice grade analog 2Rs15d were conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) to enable fast and efficient (68)Ga labeling. Biodistribution and PET/CT studies were performed on HER2-positive and -negative tumor xenografts. The effect of injected mass on biodistribution was evaluated. The biodistribution data were extrapolated to calculate radiation dose estimates for the adult female using OLINDA software. A single-dose extended-toxicity study for NOTA-2Rs15d was performed on healthy mice up to a dose of 10 mg/kg. RESULTS: Radiolabeling was quantitative (>97%) after 5 min of incubation at room temperature; specific activity was 55-200 MBq/nmol. Biodistribution studies showed fast and specific uptake (percentage injected activity [%IA]) in HER2-positive tumors (3.13 ± 0.06 and 4.34 ± 0.90 %IA/g for (68)Ga-NOTA-2Rs15dHis6 and (68)Ga-NOTA-2Rs15d, respectively, at 1 h after injection) and high tumor-to-blood and tumor-to-muscle ratios at 1 h after injection, resulting in high-contrast PET/CT images with high specific tumor uptake. A remarkable finding of the biodistribution studies was that kidney uptake was reduced by 60% for the Nanobody lacking the C-terminal His6 tag. The injected mass showed an effect on the general biodistribution: a 100-fold increase in NOTA-2Rs15d mass decreased liver uptake from 7.43 ± 1.89 to 2.90 ± 0.26 %IA/g whereas tumor uptake increased from 2.49 ± 0.68 to 4.23 ± 0.99 %IA/g. The calculated effective dose, based on extrapolation of mouse data, was 0.0218 mSv/MBq, which would yield a radiation dose of 4 mSv to a patient after injection of 185 MBq of (68)Ga-NOTA-2Rs15d. In the toxicity study, no adverse effects were observed after injection of a 10 mg/kg dose of NOTA-2Rs15d. CONCLUSION: A new anti-HER2 PET tracer, (68)Ga-NOTA-2Rs15d, was synthesized via a rapid procedure under mild conditions. Preclinical validation showed high-specific-contrast imaging of HER2-positive tumors with no observed toxicity. (68)Ga-NOTA-2Rs15d is ready for first-in-human clinical trials.