Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.

Methyl mercury and stable isotopes of nitrogen reveal that a terrestrial spider has a diet of emergent aquatic insects.

Terrestrial spiders transfer methyl mercury (MeHg) to terrestrial consumers such as birds, but how spiders become contaminated with MeHg is not well understood. In the present study, the authors used stable isotopes of nitrogen in combination with MeHg to determine the source of MeHg to terrestrial long-jawed orb weaver spiders (Tetragnatha sp). The authors collected spiders and a variety of other aquatic and terrestrial taxa from 10 shallow ponds in north Texas, USA. Based on MeHg concentrations and stable nitrogen isotope ratios, the authors identified distinct aquatic- and terrestrial-based food chains. Long-jawed orb weaver spiders belonged to the aquatic-based food chain, indicating that they are exposed to MeHg through their consumption of emergent aquatic insects. Additionally, the present study suggests that ecologists can use stable isotopes of nitrogen (δ(15) N) in conjunction with MeHg speciation analysis to distinguish between aquatic and terrestrial food chains.

Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics.

Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.

A survey of methods for the purification of microbial flavohemoglobins.

Over the past decade, the flavohemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse organisms, including yeasts and fungi but particularly pathogenic bacteria. Flavohemoglobins--the best-characterized class of microbial globin--comprise two domains: a globin domain with a noncovalently bound heme B and a flavin domain with recognizable binding sites for FAD and NAD(P)H. Hmp was first identified in Escherichia coli and now has a clearly defined role in NO biology in that organism: its synthesis is markedly up-regulated by NO, and hmp knockout mutants of E. coli and Salmonella typhimurium are severely compromised for survival in the presence of NO in vitro and in pathogenic lifestyles. In the presence of molecular O2, Hmp catalyzes an oxygenase or denitrosylase reaction in which NO is stoichiometrically converted to nitrate ion, which is relatively innocuous. In this chapter, we present a survey of the methods used to express and purify the flavohemoglobins from diverse microorganisms and describe in more detail three methods developed and used in this laboratory for the E. coli protein. Particular problems are highlighted, particularly (a) the toxic consequences of Hmp overexpression that result from its ability to catalyze partial oxygen reduction and (b) the expression of protein with substoichiometric content of redox-active flavin and heme centers.