Determination of Antibody–Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation

Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic

Additional file 3: of Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism

Supporting data. (XLSX 45.3 kb

Additional file 2: Figure S2. of Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism

Ucp3, Acadl and Cpt1b expression in C2C12 myoblasts cultured and differentiated over 6 days in the presence of 500 μM nitrate. Data are represented as mean ± SEM, n = 3 repeats per condition. *** P ≤0.001. (PDF 24 kb

Additional file 1: Figure S1. of Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism

Muscle differentiation marker expression in C2C12 myoblasts cultured and differentiated over 6 days in the presence of 0, 50 and 500 μM nitrate, and (A) in the presence and absence of sGCi (1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 1 μM) and (B) in the presence and absence of PGKi (KT5823, 1 μM). Data are represented as mean ± SEM, n = 4 repeats per condition. (PDF 52 kb

Additional file 1: of Mechanistic insights revealed by lipid profiling in monogenic insulin resistance syndromes

Supplementary Table S1 and Figures S1 and S2. (DOCX 102 kb

Microfluidic-Enabled Intracellular Delivery of Membrane Impermeable Inhibitors to Study Target Engagement in Human Primary Cells

Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure–activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes

Additional file 1: of Hepatic steatosis risk is partly driven by increased de novo lipogenesis following carbohydrate consumption

contains all the additional supplementary data in the form of a Word document. (DOCX 1130 kb