Active targeting is more effective than conventional passive targeted drug delivery systems in increasing therapeutic efficacy and minimizing systemic toxicities. Importantly, the nanoparticle-based targeted drug delivery systems combine active and passive targeting properties and significantly enhance therapeutic efficacy. In this study, we utilized ultrasmall superparamagnetic iron oxide (uSPIO) nanoparticles conjugated with anti-prostate-specific membrane antigen (PSMA) 5D3 monoclonal antibody, mertansine (DM1) antitubulin agent, and fluorophore to develop a targeted uSPIO-5D3-DM1-AF488/CF750 nanotheranostic for PSMA(+) prostate cancer (PC) therapy. This agent enables multimodality in vivo imaging using near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI). uSPIO-5D3-DM1-AF488 is selectively internalized into PSMA-positive cells by receptor-mediated endocytosis, and uSPIO-5D3-DM1-CF750 exhibited 1.62 and 166.2 ng/mL IC50 values in PSMA(+) and PSMA(-) cells, respectively. The image-guided therapeutic study was conducted in vivo in human PC xenograft mouse models bearing bilateral PSMA(±) tumors (n = 10, two 10 mg/kg doses on days 1 and 14). The therapeutic results exhibited a significant control of the growth of PSMA(+) tumors starting at day 5 (p = 0.05) and significantly improved efficacy after day 9 (p = 0.0005) during the treatment period (t = 21 days). We observed the PSMA-specific uptake of uSPIO-5D3-DM1-CF750 in tumors in NIR IVIS Xenogen images and T1- and T2-weighted MRI with 20.6% and 42% reduction of overall T1 and T2, respectively. Approximately 70% of mice with PSMA(+) tumors treated with uSPIO-5D3-DM1-CF750 survived or did not exceed the threshold level of the tumor size during the treatment. Ex vivo biodistribution study proved 50% and 45% higher uptake of uSPIO-5D3-DM1-CF750 by PSMA(+) tumors compared to untargeted uSPIO-DM1-CF750 by PSMA(+) tumors and uSPIO-5D3-DM1-CF750 by PSMA(-) tumors, respectively. ICP-MS analysis demonstrated a 73% increase in uSPIO-5D3-DM1-CF750 uptake by PSMA(+) tumors compared to PSMA(+) tumors treated with pure uSPIO. The toxicological results reveal the safe profile in systemic toxicities without life-threatening changes in the complete blood count and clinical chemistry profile of toxicology.

• Klíčová slova
• MRI, anti-PSMA antibody, image-guided drug delivery, optical imaging, prostate cancer, prostate-specific membrane antigen, targeted therapy, ultrasmall superparamagnetic iron oxide nanoparticles,
• MeSH
• antigeny povrchové * metabolismus   MeSH
• dextrany chemie   MeSH
• glutamátkarboxypeptidasa II * metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční tomografie * MeSH
• magnetické nanočástice oxidů železa * chemie   MeSH
• magnetické nanočástice * chemie   MeSH
• maytansin chemie   farmakologie   analogy a deriváty   terapeutické užití   MeSH
• monoklonální protilátky chemie   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty * farmakoterapie   diagnostické zobrazování   patologie   metabolismus   MeSH
• teranostická nanomedicína * metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové * MeSH
• dextrany MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II * MeSH
• magnetické nanočástice * MeSH
• maytansin MeSH
• monoklonální protilátky MeSH

Prostate cancer (PC) is an aggressive cancer that can progress rapidly and eventually become castrate-resistant prostate cancer (CRPC). Stage IV metastatic castrate-resistant prostate cancer (mCRPC) is an incurable late-stage cancer type with a low 5-year overall survival rate. Targeted therapeutics such as antibody-drug conjugates (ADCs) based on high-affinity monoclonal antibodies and potent drugs conjugated via smart linkers are being developed for PC management. Conjugating further with in vitro or in vivo imaging agents, ADCs can be used as antibody-theranostic conjugates (ATCs) for diagnostic and image-guided drug delivery. In this study, we have developed a novel ATC for PSMA (+) PC therapy utilizing (a) anti-PSMA 5D3 mAb, (b) Aurora A kinase inhibitor, MLN8237, and (c) for the first time using tetrazine (Tz) and trans-cyclooctene (TCO) click chemistry-based conjugation linker (CC linker) in ADC development. The resulting 5D3(CC-MLN8237)3.2 was labeled with suitable fluorophores for in vitro and in vivo imaging. The products were characterized by SDS-PAGE, MALDI-TOF, and DLS and evaluated in vitro by optical imaging, flow cytometry, and WST-8 assay for cytotoxicity in PSMA (+/-) cells. Therapeutic efficacy was determined in human PC xenograft mouse models following a designed treatment schedule. After the treatment study animals were euthanized, and toxicological studies, complete blood count (CBC), blood clinical chemistry analysis, and H&E staining of vital organs were conducted to determine side effects and systemic toxicities. The IC50 values of 5D3(CC-MLN8237)3.2-AF488 in PSMA (+) PC3-PIP and PMSA (-) PC3-Flu cells are 8.17 nM and 161.9 nM, respectively. Pure MLN8237 shows 736.9 nM and 873.4 nM IC50 values for PC3-PIP and PC3-Flu cells, respectively. In vivo study in human xenograft mouse models confirmed high therapeutic efficacy of 5D3(CC-MLN8237)3.2-CF750 with significant control of PSMA (+) tumor growth with minimal systemic toxicity in the treated group compared to PSMA (-) treated and untreated groups. Approximately 70% of PSMA (+) PC3-PIP tumors did not exceed the threshold of the tumor size in the surrogate Kaplan-Meyer analysis. The novel ATC successfully controlled the growth of PSMA (+) tumors in preclinical settings with minimal systemic toxicities. The therapeutic efficacy and favorable safety profile of novel 5D3(CC-MLN8237)3.2 ATC demonstrates their potential use as a theranostic against aggressive PC.

• Klíčová slova
• Aurora A kinase inhibition, MLN8237, PSMA -prostate-specific membrane antigen, antibody-theranostic conjugates, image-guided drug delivery, prostate cancer, targeted therapy, theranostics,
• Publikační typ
• časopisecké články MeSH

Prostate cancer (PCa) tops the list of cancer-related deaths in men worldwide. Prostate-specific membrane antigen (PSMA) is currently the most prominent PCa biomarker, as its expression levels are robustly enhanced in advanced stages of PCa. As such, PSMA targeting is highly efficient in PCa imaging as well as therapy. For the latter, PSMA-positive tumors can be targeted directly by using small molecules or macromolecules with cytotoxic payloads or indirectly by engaging the immune system of the host. Here we describe the engineering, expression, purification, and biological characterization of bispecific T-cell engagers (BiTEs) that enable targeting PSMA-positive tumor cells by host T lymphocytes. To this end, we designed the 5D3-αCD3 BiTE as a fusion of single-chain fragments of PSMA-specific 5D3 and anti-CD3 antibodies. Detailed characterization of BiTE was performed by a combination of size-exclusion chromatography, differential scanning fluorimetry, and flow cytometry. Expressed in insect cells, BiTE was purified in monodisperse form and retained thermal stability of both functional parts and nanomolar affinity to respective antigens. 5D3-αCD3's efficiency and specificity were further evaluated in vitro using PCa-derived cell lines together with peripheral blood mononuclear cells isolated from human blood. Our data revealed that T-cells engaged via 5D3-αCD3 can efficiently eliminate tumor cells already at an 8 pM BiTE concentration in a highly specific manner. Overall, the data presented here demonstrate that the 5D3-αCD3 BiTE is a candidate molecule of high potential for further development of immunotherapeutic modalities for PCa treatment.

• Publikační typ
• časopisecké články MeSH

Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.

• MeSH
• antigeny povrchové chemie   metabolismus   MeSH
• buňky PC-3 MeSH
• endocytóza MeSH
• fluorescenční spektrometrie metody   MeSH
• glutamátkarboxypeptidasa II chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sondy chemie   metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty diagnóza   metabolismus   MeSH
• optické zobrazování metody   MeSH
• proteinové domény MeSH
• transplantace heterologní MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• molekulární sondy MeSH

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

• Klíčová slova
• NAALADase, antibody fragment, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer, prostate-specific membrane antigen,
• MeSH
• antigeny povrchové imunologie   MeSH
• buněčné linie MeSH
• buňky PC-3 MeSH
• fluorescence MeSH
• glutamátkarboxypeptidasa II imunologie   MeSH
• hmyz MeSH
• jednořetězcové protilátky imunologie   MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty imunologie   MeSH
• rekombinantní proteiny imunologie   MeSH
• xenogenní modely - testy protinádorové aktivity metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• jednořetězcové protilátky MeSH
• monoklonální protilátky MeSH
• rekombinantní proteiny MeSH