An accelerated, clinical-grade protocol to generate high yields of type 1-polarizing messenger RNA-loaded dendritic cells for cancer vaccination.

BACKGROUND: Many efforts have been devoted to improve the performance of dendritic cell (DC)-based cancer vaccines. Ideally, a DC vaccine should induce robust type 1-polarized T-cell responses and efficiently expand antigen (Ag)-specific cytotoxic T-cells, while being applicable regardless of patient human leukocyte antigen (HLA) type. Production time should be short, while maximally being good manufacturing practice (GMP)-compliant. We developed a method that caters to all of these demands and demonstrated the superiority of the resulting product compared with DCs generated using a well-established "classical" protocol. METHODS: Immunomagnetically purified monocytes were cultured in a closed system for 3 days in GMP-compliant serum-free medium and cytokines, and matured for 24 h using monophosphoryl lipid A (MPLA)+ interferon-gamma (IFN-γ). Mature DCs were electroporated with messenger RNA (mRNA) encoding full-length antigen and cryopreserved. "Classical" DCs were cultured for 8 days in flasks, with one round of medium and cytokine supplementation, and matured with tumor necrosis factor alpha (TNF-α) + prostaglandin E2 (PGE2) during the last 2 days. RESULTS: Four-day MPLA/IFN-γ-matured DCs were superior to 8-day TNF-α/PGE2-matured DCs in terms of yield, co-stimulatory/co-inhibitory molecule expression, resilience to electroporation and cryopreservation and type 1-polarizing cytokine and chemokine release after cell thawing. Electroporated and cryopreserved DCs according to our protocol efficiently present epitopes from tumor antigen-encoding mRNA, inducing a strong expansion of antigen-specific CD8+ T-cells with full cytolytic capacity. CONCLUSION: We demonstrate using a GMP-compliant culture protocol the feasibility of generating high yields of mature DCs in a short time, with a superior immunogenic profile compared with 8-day TNF-α/PGE2-matured DCs, and capable of inducing vigorous cytotoxic T-cell responses to antigen from electroporated mRNA. This method is now being applied in our clinical trial program.

Possible nitrosodimethylamine formation in comparative in vitro nitrosation experiments with six different tetracycline antibiotics.

The hydrochlorides of tetracycline, minocycline, chlorotetracycline, anhydrochlorotetracycline, desmethylchlorotetracycline, oxytetracycline and doxycycline were reacted with sodium nitrite for two hours at 37 degrees C in aqueous buffer solutions at pH 2 and 4 under simulated stomach conditions. NDMA formation was detected from minocycline, doxycycline, oxytetracycline and anhydrochlorotetracycline by GLC and GLC-MS analysis. NDMA formation from minocycline and dodxycycline was blocked by ascorbic acid. The catalytic effect of sodium thiocyanate for NDMA formation from minocycline and nitrite was investigated. The different reactivities of the tested tetracyclines towards nitrite in acidic solutions (NDMA formation from minocycline, doxycycline, oxytetracycline and no NDMA formation from tetracycline, chlorotetracycline and desmethylchlorotetracycline) may be understood from stereochemical considerations. The failure of dealkylative nitrosation reactions in the latter tetracyclines is explained by the formation of intramolecular hydrogen bridge linkages between epidimethylamino groups at C-4 and OH groups at C-6. This hypothesis was proved by the observations of NDMA formation from anhydrochlortetracycline and sodium nitrite at pH 2.

[On the problem of dimethylnitrosamine formation from tetracycline-derivatives by nitrosation reaction in acidic medium (author's transl)]. / Zur Frage der Bildung von Dimethylnitrosamin aus Tetracyclin-Derivaten bei der Nitrosierung in saurer Lösung.

The hydrochlorides from tetracycline and six tetracycline derivatives -- 7-dimethylamino-6-des-methyl-6-desoxytetracycline [minocycline], 7-chlorotetracycline [chlorotetracycline], 7-chloro-anhydrotetracycline [anhydrochlorotetracycline, 7-chloro-6-desmethyltetracycline [demethylchlorotetracycline], 5-hydroxytetracycline [oxytetracycline] and 6-desoxy-5-hydroxytetracycline [doxycycline] -- were reacted with different amounts of sodium nitrite at 37 degrees C for two hours in aqueous buffer solutions at pH 2 and 4. Dimethylnitrosamine formation was confirmed by gas-liquid chromatography and by combined gas-liquid chromatography/mass-spectrometry from minocycline, doxycycline, oxytetracycline and anhydrochlorotetracycline. Dimethylnitrosamine formation from minocycline and doxycycline was blocked by ascorbic acid. The catalytic effect of sodium thiocyanate for the dimethylnitrosamine formation from minocycline and nitrite was investigated. The different reactivity of the investigated tetracycline derivatives towards nitrite in acidic solutions is discussed by stereochemical considerations in connection with the formation of hydrogen bridge linkages. This hypothesis was confirmed by dimethylnitrosamine formation from anhydrochlorotetracycline and sodium nitrite at pH 2.

Mass spectrometry of nitrosamines: rearrangement of nitrosobenzylmethylamine molecular ions with loss of OH-radicals investigated by deuterated derivatives.

Nitrosamines (R1R2NO) containing a certain R groups yield mass spectra with M+-OH fragments which can be accounted for by intramolecular hydrogen shift with subsequent cleavage of OH-radicals. As can be shown with mass spectra of specifically deuterated derivatives of nitrosobenzylmethylamine - nitrosobenzyltrideuteriomethylamine, alpha,alpha-nitrosodideuteriobenzylmethylamine and o,o',p-nitrosotrideuteri0-benzylmethylamine - hydrogen transfer within the molecular ion of nitrosobenzylmethylamine occurs selectively onto the oxygen of the nitroso group from the benzylethylene group, probably via a five-membered cyclic transition state. Cleavage of OH radicals leads to formation of M+-17 ions, the stability of which can be explained by mesomerically stabilized cyclic diazirinium ions.