High-Throughput IgG Reformatting and Expression Using Hybrid Secretion Signals and InTag Positive Selection Technology.

We have previously published protocols for high-throughput IgG reformatting and expression, that enable rapid reformatting of phage-displayed antibody Fab fragments into a single dual expression vector for full IgG expression in Expi293F cells (Chen et al. Nucleic Acids Res 42:e26, 2014; Chen et al. Methods in Molecular Biology, vol 1701, 2018). However, when working with phage clones from a naïve library containing highly diverse N-terminal sequences, where the 5' PCR primers bind, the PCR step can become cumbersome. To overcome this limitation, we have investigated and found that the C-terminal 7 amino acid residues of the human antibody VH1 secretion signal can be replaced with those from ompA or pelB bacterial signals to form hybrid signal sequences that can drive strong IgG expression in Expi293F cells. The use of such hybrid signals allows any Fab fragment in the library to be amplified and cloned into the IgG expression vector using only a single 5' PCR primer targeting the bacterial secretion signal of the light or heavy chain, thus dramatically simplifying the IgG reformatting workflow.

Optimizing high throughput antibody purification by using continuous chromatography media.

The ability to engineer monoclonal antibodies (mAbs) with high specificity made mAbs the fastest growing segment in the drug market. mAbs represent 8 of the top 20 selling drugs with combined sales of more than 57 billion US$ per year. The ability to purify large numbers of mAbs with sufficient yields for initial screening campaigns has direct impact on the timelines of a project. Automated liquid handling (ALH)-based mAb purification platforms have been used to facilitate the production of large numbers of mAbs. However, the ongoing pressure to de-risk potential lead molecules at an early development stage by including bio-physical characterization of mAbs has further increased the demand to produce sufficient quantities from limited sample volumes. A bottleneck so far has been the limited dynamic binding capacity of these systems, which is partly due to the binding properties of commonly used Protein A affinity matrices. The present publication suggests that by using a Protein A matrix optimized for continuous chromatography applications the yields of ALH-based but also standard lab-scale mAb purifications can be significantly increased without the need to change established protocols.

High-Throughput IgG Reformatting and Expression.

We have recently described a one-step zero-background IgG reformatting method that enables the rapid reformatting of phage-displayed antibody fragments into a single-mammalian cell expression vector for full IgG expression (Chen et al. Nucleic Acids Res 42:e26, 2014). The strategy utilizes our unique positive selection method, referred to as insert-tagged (InTag) positive selection, where a positive selection marker (e.g. chloramphenicol-resistance gene) is cloned together with the antibody inserts into the expression vector. The recombinant clones containing the InTag adaptor are then positively selected without cloning background, thus bypassing the need to plate out cultures and screen colonies. This IgG reformatting method is rapid and can be automated and performed in a high-throughput (HTP) format. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated. We have further optimized the protocol for IgG reformatting since the initial publication of this method (Chen et al. Nucleic Acids Res 42:e26, 2014) and also updated the transient transfection protocol using Expi293F cells, which are described herein.

Production of monoclonal antibodies reactive with ovine eosinophils.

BACKGROUND: There is strong evidence implicating eosinophils in host defence against parasites as well as allergic disease pathologies. However, a lack of reagents such as monoclonal antibodies (mAbs) specific for eosinophils has made it difficult to confirm the functional role of eosinophils in such disease conditions. Using an established mammary model of allergic inflammation in sheep, large numbers of inflammatory cells enriched for eosinophils were collected from parasite-stimulated mammary glands and used for the generation of mAbs against ovine eosinophils. RESULTS: A panel of mAbs was raised against ovine eosinophils of which two were shown to be highly specific for eosinophils. The reactivity of mAbs 3.252 and 1.2 identified eosinophils from various cell and tissue preparations with no detectable reactivity on cells of myeloid or lymphoid lineage, tissue mast cells, dendritic cells, epithelial cells or other connective tissues. Two other mAbs generated in this study (mAbs 4.4 and 4.10) were found to have reactivity for both eosinophils and neutrophils. CONCLUSION: This study describes the production of new reagents to identify eosinophils (as well as granulocytes) in sheep that will be useful in studying the role of eosinophils in disease pathologies in parasite and allergy models.