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.

Linker technologies for antibody-drug conjugates.

Antibody-drug conjugates (ADCs), which combine the specificity, favorable pharmacokinetics, and biodistribution of a monoclonal antibody (mAb) with the cytotoxic potency of a drug, are promising new therapies for cancer. Along with the development of monoclonal antibodies (mAbs) and cytotoxic drugs, the design of the linker is of essential importance, because it impacts the efficacy and tolerability of ADCs. The linker needs to provide sufficient stability during systemic circulation but allow for the rapid and efficient release of the cytotoxic drug in an active form inside the tumor cells. This review provides an overview of linker technologies currently used for ADCs and advances that have resulted in linkers with improved properties. Also provided is a brief summary of some considerations for the conjugation of antibody and drug linker such as drug-to-antibody ratio and site of conjugation.

Synthesis of rhamnogalacturonan I fragments by a modular design principle.

The improved syntheses of methyl 2-O-acetyl-3-O-benzyl-alpha-L-rhamnopyranoside (12) and 1,2-di-O-acetyl-3-O-benzyl-alpha-L-rhamnopyranose (15), which were used as glycosyl acceptor and donor, respectively, are described. Glycosylation of the O-4 position of both rhamnose derivatives with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide (26) provided disaccharides 27 and 29. After partial deprotection of 27 and coupling of the resulting 28 with disaccharide 19, tetrasaccharide 31 was obtained. Furthermore, transforming of 29 into the corresponding bromide 30 and coupling with galacturonates 16 and 32 provided trisaccharides 33 and 34, respectively, which could be regarded as building blocks of ramified rhamnogalacturonan fragments. The preparation of tetra- (21) and hexasaccharide (25) of rhamnogalacturonan I is reported to demonstrate the feasibility of the synthesis of larger pectin fragments using the modular design principle with this type of building blocks.

A nanoengineering approach for investigation and regulation of protein immobilization.

It is known that protein attachment to surfaces depends sensitively upon the local structure and environment of the binding sites at the nanometer scale. Using nanografting and reversal nanografting, both atomic force microscopy (AFM)-based lithography techniques, protein binding sites with well-defined local environments are designed and engineered with nanometer precision. Three proteins, goat antibiotin immunoglobulin G (IgG), lysozyme, and rabbit immunoglobulin G, are immobilized onto these engineered surfaces. Strong dependence on the dimension and spatial distribution of protein binding sites are revealed in antibody recognition, covalent attachment via primary amine residues and surface-bound aldehyde groups. This investigation indicates that AFM-based nanolithography enables the production of protein nanostructures, and more importantly, protein-surface interactions at a molecular level can be regulated by changing the binding domains and their local environment at nanometer scale.

The structure of lipid A of the lipopolysaccharide from Burkholderia caryophylli with a 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residue exclusively in glycosidic linkage.

From the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli, the complete structure of lipid A has been characterized. For the first time, a 4-amino-4-deoxy-L-arabinopyranose 1-phosphate residue was proven to be exclusively linked to the reducing end of lipid A from a wild-type LPS. The LPSs of B. caryophylli were degraded by mild acetate buffer hydrolysis at pH 4.4. The obtained lipid A was analyzed as such, and also after de-O-acylation or dephosphorylation. The structure of lipid A was identified mainly by means of matrix-assisted laser desorption/ionisation mass spectrometry, and by various 1D and 2D (1)H and (13)C NMR spectroscopic measurements.