Cysteine metabolic engineering and selective disulfide reduction produce superior antibody-drug-conjugates.

Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.

Impacts of fast production of afucosylated antibodies and Fc mutants in ExpiCHO-S™ for enhancing FcγRIIIa binding and NK cell activation.

This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG1 and murine IgG2a generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.

Mercury in fish from the Madeira River and health risk to Amazonian and riverine populations.

The objective of this study was to quantify total mercury in highly popular Amazonian fish pacu, curimatã, jaraqui, and sardinha from the Madeira River and to estimate the exposure to methylmercury from fish consumption. The samples were obtained from two locations - Puruzinho Igarapé and Santa Rosa - near Humaitá, Amazonia, Brazil in two seasons of 2015 (high and low waters). The fish were identified, weighed and measured, and lipids were quantified. Total mercury was determined by gold amalgamation-atomic absorption spectrometry. Mean levels were used to calculate exposure of Amazonian and riverine populations. There was significant correlation (p < 0.05) between length × weight for all fish; length × lipid and weight × lipid were significant only for pacu. Total mercury levels varied along muscle tissue for the fish, except for sardinha; therefore muscle from the dorsal area along the fish were sampled, homogenized and used for analysis. The levels of total mercury varied from 0.01 to 0.46 mg/kg, with higher median levels in sardinha (0.24 mg/kg), followed by curimatã (0.16 mg/kg), jaraqui (0.13 mg/kg) and pacu (0.04 mg/kg), corresponding with the respective feeding habits along the trophic chain. Total mercury levels were not affected by the location of fish capture and by high and low waters seasons. Total mercury correlated significantly with length and weight for jaraqui and with length for sardinha (negative correlation). Total mercury levels in fish complied with legislation; however, exposures to methylmercury from fish consumption overpassed the safe intake reference dose for sardinha for Amazonians; however, for the riverine communities, all of the fish would cause potential health risk, mainly for children and women of childbearing age.

[Portuguese recommendations for pain management by pharmocotherapy in inflammatory arthritis]. / Recomendações portuguesas para o tratamento e controlo farmacológico da dor nas artropatias inflamatórias.

OBJECTIVES: To develop Portuguese evidence-based recommendations for pain management by pharmocotherapy in inflammatory arthritis. METHODS: The Portuguese project was integrated in the multinational 3E Initiative (Evidence, Expertise, Exchange) 2010 where a total of 453 rheumatologists from 17 countries have participated. The clinical questions concerning pain were formulated and the Portuguese group added 2 more questions. A systematic literature search was performed in Medline, Embase, Cochrane Library and 2008-2009 EULAR and ACR abstracts. The selected articles were systematically reviewed and the evidence was defined according to the Oxford Levels of Evidence. In each country a group of experts joined to discuss their national recommendations. In Portugal, the national meeting was held in October 2010, where 33 rheumatologists discussed and voted by Delphi method the national recommendations. Finally, the agreement among the rheumatologists and the potential impact on their clinical practice was assessed. RESULTS: Thirteen national recommendations were formulated: pain measure scores; analgesic combination therapy; pharmacotherapy in preconception, pregnancy and lactation periods; pharmacotherapy according to comorbilities; safety of NSAIDs and/or paracetamol with methotrexate combination therapy; efficacy and safety of continuous/on-demand NSAIDs; opioids, paracetamol, corticosteroids, antidepressants, neuromodulators and muscle relaxants role and effectiveness; risk factors for the development of chronic pain and the role of topic analgesics. CONCLUSION: The portuguese recommendations for the pain management by pharmacotherapy in inflammatory arthritis were formulated according to the best evidence and supported by a panel of 63 rheumatologists. The differences between the national and international recommendations are reported in this article.