Site-specific conjugation of native antibody.

Traditionally, non-specific chemical conjugation, such as acylation of amines on lysine or alkylation of thiols on cysteines, are widely used; however, they have several shortcomings. First, the lack of site-specificity results in heterogeneous products and irreproducible processes. Second, potential modifications near the complementarity determining region (CDR) may reduce binding affinity and specificity. Conversely, site-specific methods produce well-defined and more homogenous antibody conjugates, ensuring developability and clinical applications. Moreover, several recent side-by-side comparisons of site-specific and stochastic methods have demonstrated that site-specific approaches are more likely to achieve their desired properties and functions, such as increased plasma stability, less variability in dose-dependent studies (particularly at low concentrations), enhanced binding efficiency, as well as increased tumor uptake. Herein we review several standard and practical site-specific bioconjugation methods for native antibodies, i.e., those without recombinant engineering. First, chemo-enzymatic techniques, namely transglutaminase (TGase)-mediated transamidation of a conserved glutamine residue and glycan remodeling of a conserved asparagine N-glycan (GlyCLICK), both in the Fc region. Second, chemical approaches such as selective reduction of disulfides (ThioBridge) and N-terminal amine modifications. Furthermore, we list site-specific antibody-drug conjugates (ADCs) in clinical trials along with the future perspectives of these site-specific methods.

Hydration status affects thirst and salt preference but not energy intake or postprandial ghrelin in healthy adults: A randomised crossover trial.

BACKGROUND: Few studies have investigated the effect of hydration status on appetite for food in healthy adults. Prior work suggests hydration status does not alter appetite or energy intake, with mixed findings regarding appetite hormone secretion. However, an extensive investigation into both the psychological and physiological appetitive responses to hydration status has never been conducted. OBJECTIVE: To investigate the effect of hydration status on multiple facets of appetite. DESIGN: After 3 days pre-trial standardization, a range of appetite tasks were conducted when hypohydrated (HYPO) and euhydrated (EUHY) in 16 healthy participants (8 men). Hydration status was manipulated via dehydration in a heat tent for 60 min and subsequent fluid restriction (HYPO) or replacement (EUHY). The next day, a food reward computer task was completed followed by an ad libitum pasta meal. Pre- and post-prandial visual analogue scales assessing hunger, fullness, and flavour desires (sweet, salty, savoury and fatty) were additionally completed. Blood samples were taken the previous day before the hydration interventions in a euhydrated state, and in the fasted and post-prandial state during HYPO and EUHY. RESULTS: HYPO induced -1.9 ±â€¯1.2% body mass change, compared to -0.2 ±â€¯0.6% , with accompanying changes in markers of hypohydration which were not seen during EUHY. A higher desire for foods was associated with a higher water content but the association was weaker in EUHY compared to HYPO, (ß= -0.33 mm/g of food water content, p < 0.001) in the food reward task. Visual analogue scales showed similar hunger and fullness between interventions, but during HYPO there was consistently higher thirst (average range in difference 27-32 mm across all time points) and lower fasted desire for salt (-23, 95% CI -10, -35 mm). Ad libitum energy intake (HYPO 1953 ±â€¯742 kJ, EUHY 2027 ±â€¯926 kJ; p = 0.542) and post-prandial ghrelin concentrations (HYPO 180 ±â€¯65 pg mL-1, EUHY 188 ±â€¯71 pg mL-1; p = 0.736) were similar by hydration status. CONCLUSIONS: An acute manipulation to hydration status altered desire for salt and foods of differing water contents, but did not influence energy intake at an ad libitum pasta meal. Further research should investigate whether these appetites would alter food choice.

Effect of acute hypohydration on glycemic regulation in healthy adults: a randomized crossover trial.

The aim of this study was to investigate the acute effect of hydration status on glycemic regulation in healthy adults and explore underlying mechanisms. In this randomized crossover trial, 16 healthy adults (8 men, 8 women) underwent an oral glucose tolerance test (OGTT) when hypohydrated and rehydrated after 4 days of pretrial standardization. One day before OGTT, participants were dehydrated for 1 h in a heat tent with subsequent fluid restriction (HYPO) or replacement (RE). The following day, an OGTT was performed with metabolic rate measurements and pre- and post-OGTT muscle biopsies. Peripheral quantitative computer tomography thigh scans were taken before and after intervention to infer changes in cell volume. HYPO (but not RE) induced 1.9% (SD 1.2) body mass loss, 2.9% (SD 2.7) cell volume reduction, and increased urinary hydration markers, serum osmolality, and plasma copeptin concentration (all P ≤ 0.007). Fasted serum glucose [HYPO 5.10 mmol/l (SD 0.42), RE 5.02 mmol/l (SD 0.40); P = 0.327] and insulin [HYPO 27.1 pmol/l (SD 9.7), RE 27.6 pmol/l (SD 9.2); P = 0.809] concentrations were similar between HYPO and RE. Hydration status did not alter the serum glucose ( P = 0.627) or insulin ( P = 0.200) responses during the OGTT. Muscle water content was lower before OGTT after HYPO compared with RE [761 g/kg wet wt (SD 13) vs. 772 g/kg wet wt (SD 18) RE] but similar after OGTT [HYPO 779 g/kg wet wt (SD 15) vs. RE 780 g/kg wet wt (SD 20); time P = 0.011; trial × time P = 0.055]. Resting energy expenditure was similar between hydration states (stable between -1.21 and 5.94 kJ·kg-1·day-1; trial P = 0.904). Overall, despite acute mild hypohydration increasing plasma copeptin concentrations and decreasing fasted cell volume and muscle water, we found no effect on glycemic regulation. NEW & NOTEWORTHY We demonstrated for the first time that an acute bout of hypohydration does not impact blood sugar control in healthy adults. Physiological responses to mild hypohydration (<2% body mass loss) caused an elevation in copeptin concentrations similar to that seen in those with diabetes as well as reducing cell volume by ~3%; both of these changes had been hypothesized to cause a higher blood sugar response.