Small bispecific antibodies that induce T-cell–mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell–recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 103-times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.

Extracellular-superoxide dismutase (EC-SOD), one of the SOD isozymes, is negatively regulated under hypoxic conditions, and decreases in its expression may exacerbate vascular diseases. Moreover, epigenetics, such as DNA methylation and histone modifications, are known to play a critical role in the progression of cancer, type 2 diabetes, and atherosclerosis. We previously investigated the involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK) in decreases in EC-SOD expression in hypoxic COS7 cells; however, the role of epigenetics in this process currently remains unknown. In the present study, we demonstrated that the hypoxia mimetic cobalt chloride (CoCl 2 ) decreased histone acetylation levels, and a pretreatment with 4-phenyl butyric acid (PBA), an inhibitor of histone deacetylase, significantly suppressed CoCl 2 -elicited histone deacetylation and decreases in EC-SOD. We found that CoCl 2 -elicited decreases in EC-SOD were accompanied by reductions in histone H3 acetylation levels within its promoter region. Furthermore, luteolin, a well-known flavonoid, significantly suppressed the CoCl 2 -elicited accumulation of ROS, p38-MAPK activation, and histone deacetylation. Collectively, the results of the present study showed for the first time that CoCl 2 decreases the expression of EC-SOD through its deacetylation and luteolin may be one of the seed compounds that maintain redox homeostasis, even under hypoxic conditions.

Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues—which can be chemical conjugation sites—were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

T his study qualitatively assessed the impact of colocating hospitalists and nurses on patient care. 1 Using focus groups, nurse and provider participants reported a positive impact, mediated by proximity and improved communication. Focus groups also developed a conceptual model demonstrating how geographic localization could affect several key elements of patient care.This paper also elucidated four potential unintended consequences of co-localization: 1) increased communication interruptions, which could threaten patient safety; 2) rather than a constant flow of new patients from multiple teams, co-located teams had admission flow problems, with patients often arriving in boluses, which tended to overwhelm nursing resources; 3) the teams were generalist teams and this could potentially disrupt wards with nursing units that are specialized; and 4) there was the potential for perverse incentives to increase length of stay, as found in previous work. 2 This study has several limitations. The sample size was small, including only two hospitalist teams and one nursing unit, and many constituents of medical teams, such as pharmacists, case managers and social workers, were not included. Moreover, despite the primary interest in the impact on patient care, there were no patients' voices reflected in the results. Despite these limitations, the model looks reasonable and fits well with what we imagine would happen if this co-localization were deployed. For the next step, researchers might want to further refine their conceptual model by more qualitative research with a larger, more diverse sample including staff other than doctors and nurses, incorporating the patient's perspective.In addition, further quantitative investigations on the impact of co-localization are also needed. Previous research of co-localization of hospitalist with nursing teams found increased satisfaction and no impact on readmission rates, 2,3 but no improvement in agreement between nurses and providers on the care plan 2 and possibly increased length of stay. 3 Research on uninvestigated domains of quality such as patient safety is also a future task.Finally, ward teams provide functions other than patient care; for example, education. The potential impact of colocalization on aspects of the medical enterprise other than direct patient care should be evaluated.

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