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NQO1 and NQO2 are closely related quinone reductases, which are FAD-linked enzymes that catalyze the 2-electron reduction of quinones. Whereas NQO1 has a well-defined role in cellular detoxification of quinones, NQO2’s function is less clear; it uses the conventional cofactors NADH/NADPH inefficiently compared to smaller nicotinamide cofactors, which are often present in low cellular amounts. This unique cofactor specificity suggests that NQO2 may have non-enzymatic functions, such as a role in intracellular signalling. The goals of this research project include: 1) examining whether cofactor specificity is conserved in amniote NQO2 enzymes and 2) re-constructing ancient enzymes along the evolutionary pathway of NQO1 and NQO2 to determine the molecular origins of cofactor specificity differences. The reductive half-reactions of H.sapiens, A.platyrhynchos, and A.mississippiensis NQO2 with NADH were investigated using stopped-flow apparatus. With increasing NADH concentration, the observed rate constant for NQO2 reduction for all three amniotes increased before reaching saturation. This indicates that the enzyme-substrate complex for NQO2 reduction exists in rapid equilibrium, leading to the conclusion of non-productive NADH binding across all amniote NQO2s. To examine the evolution of NQO1 and NQO2, a phylogenetic tree was created using amino acid sequences of numerous species. While the characterization of ancient enzyme kinetics is still ongoing, the successful expression of various ancestral proteins confirmed our analysis of the phylogeny and predicted sequences.

L-phenylalanine, L-tyrosine and L-tryptophan are aromatic amino acids that are widely used in industrial, agricultural and pharmaceutical applications. Currently, heterotrophic microorganisms are fed require an organic source of carbon to produce amino acids, photosynthetic bacteria are investigated. Mutagenesis by methyl methanesulfonate followed by selection on aromatic amino acid analogues produced mutants that are able to overproduce aromatic amino acids. Two mutant strains were investigated: TA1, which is a better L-phenylalanine overproducer, and 5FT1, which overproduces L-tryptophan. To test the influence of growth conditions on amino acid production, we grew mutant strains of Synechocytis sp. 6803 under enhanced CO2 or glucose-rich conditions. We quantified the amount of each aromatic amino acid synthesized intracellularly and transported to extracellular medium by high performance liquid chromatography. We analysed the transient production of the aromatic amino acids to see if it is a growth related process. If it is growth related, the recommended growth method would be a fed batch reactor or a turbidostat to keep the cells at the exponential phase. If it is not growth related, we would recommend to use a batch reactor to allow cells reach stationary phase and harvest after maximum production.

Antibody-drug conjugates (ADCs) have experienced a surge in clinical approvals in the past five years. Despite this success, a major limitation to ADC efficacy in solid tumors is poor tumor penetration, which leaves many cancer cells untargeted. Increasing antibody doses or co-administering ADC with an unconjugated antibody can improve tumor penetration and increase efficacy when target receptor expression is high. However, it can also reduce efficacy in low-expression tumors where ADC delivery is limited by cellular uptake. This creates an intrinsic problem because many patients express different levels of target between tumors and even within the same tumor. Here, we generated High-Avidity, Low-Affinity (HALA) antibodies that can automatically tune the cellular ADC delivery to match the local expression level. Using HER2 ADCs as a model, HALA antibodies were identified with the desired HER2 expression-dependent competitive binding with ADCs in vitro . Multi-scale distribution of trastuzumab emtansine and trastuzumab deruxtecan co-administered with the HALA antibody were analyzed in vivo , revealing that the HALA antibody increased ADC tumor penetration in high-expression systems with minimal reduction in ADC uptake in low-expression tumors. This translated to greater ADC efficacy in immunodeficient mouse models across a range of HER2 expression levels. Furthermore, Fc-enhanced HALA antibodies showed improved Fc-effector function at both high and low expression levels and elicited a strong response in an immunocompetent mouse model. These results demonstrate that HALA antibodies can expand treatment ranges beyond high expression targets and leverage strong immune responses., Competing Interests: Conflicts of Interest P.M.T. is a member of the scientific advisory boards for Nabla Bio, Adimab, Aureka Biotechnologies, and Dualitas Therapeutics. G.M.T. has served on the scientific advisory boards of AstraZeneca/Medimmune, Advanced Proteome Therapeutics, Catalent, Merck, Mersana, and Neoleukin. The authors have intellectual property related to the use of affinity-modulated antibodies for use with ADCs.