In vivo study of radioprotective effect of NO-synthase inhibitors and acetyl-L-carnitine

In vivo study of radiprotective effect of NO-synthase inhibitors and acetyl-L-carnitine This study investigated the protective effect of two nitric oxide synthase inhibitors N(omega)-Nitro-Larginine methyl ester (L-NAME, 100 mg/kg i.p.) and aminoguanidine (AG, 400 mg/kg i.p.), and an antioxidant acetyl-L-carnitine (ALC, 250 mg/kg i.p., once daily for five days) against radiation-induced damage in Wistar rats. Blood samples were collected 6 hrs after whole-body irradiation with 8 Gy. Plasma concentrations of nitrite+nitrate (NOx) and malondialdehyde (MDA) were measured by highperformance liquid chromatography. A single injection of L-NAME one hour before exposure effectively prevented the radiation-induced elevation of plasma NOx and it reduced 2.6-fold the risk for death during the subsequent 30-day period. Pretreatment with ALC prevented the radiation-induced increase in plasma MDA and it had similar effect on mortality as L-NAME did. Presumably due to its short half-life, the partially iNOS-selective inhibitor and antioxidant AG given in a single dose before exposure did not attenuate MDA and NOx and it failed to significantly improve the 30-day survival. In conclusion, pretreatment with both the nonspecific NOS inhibitor L-NAME and the antioxidant ALC markedly reduce mortality to radiation sickness in rats. The radioprotective effect may be directly related to effective attenuation of the radiation-induced elevation of NO production by L-NAME and of oxidative stress by ALC. Key words: acetyl-L-carnitine; aminoguanidine; ionizing radiation; N(omega)-Nitro-L-arginine methyl este

Targeting Prostate Cancer Using Bispecific T‑Cell Engagers against Prostate-Specific Membrane Antigen

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

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

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