Development of a new genotype–phenotype linked antibody screening system

Reviewer #1 (Public Review):

Summary:

This paper by Watanabe et al described an expression system that can express the paired heavy and light chains of IgG antibodies from single cell B cells. In addition, they used FACS sorting for specific antigens to screen/select the specific populations for more targeted cloning of mAb genes. By staining with multiple antigens, they were able to zoom in to cross-reactive antibodies.

Strengths:

A highly efficient process that combines selection/screening with dua expression of both antibody chains. It is particularly suitable for the isolation of cross-reactive antibodies against conserved epitopes of different antigens, such as surface proteins of related viruses.

Weaknesses:

(1) The overall writing is very difficult to follow and the authors need to work on significant re-writing.

(2) The paper in its current form really lacks detail and it is NOT possible for readers to repeat or follow their methods. For example: a) It is not clear whether the authors checked the serum to see if the mice were producing antibodies before they sacrificed them to harvest spleen/blood i.e. using ELISA? b) How long after administration of the second dose were the mice sacrificed? c) What cell types are taken for single B cell sorting? Splenocytes or PBMC? These are just some of the questions which need to be addressed.

(3) According to the authors, 77 clones were sorted from the PR8+ and H2+ double positive quadrant. It is surprising that after transfection and re-analysing of bulk antibody presenting EXPI cells on FACS, only 13 clones (or 8 clones? - unclear) seemed to be truly cross-reactive. If that is the case, the approach is not as efficient as the authors claimed.

Reviewer #2 (Public Review):

Summary:

Watanabe, Takashi, et al. investigated the use of the Golden Gate dual-expression vector system to enhance the modern standard for rapid screening of recombinant monoclonal antibodies. The presented data builds upon modern techniques that currently use multiple expression vectors to express heavy and light chain pairs. In a single vector, they express the linked heavy and light chain variable genes with a membrane-bound Ig which allows for rapid and more affordable cell-based screening. The final validation of H1 and H2 strain influenza screening resulted in 81 "H1+", 48 "H2+", and 9 "cross" reactive clones. The kinetics of some of the soluble antibodies were tested via SPR and validated with a competitive inhibition with classical well-characterized neutralizing clones.

Strengths:

In this study, Watanabe, Takashi, et al. further develop and refine the methodologies for the discovery of monoclonal antibodies. They elegantly merge newer technologies to speed up turnaround time and reduce the cost of antibody discovery. Their data supports the feasibility of their technique.

This study will have an impact on pandemic preparedness and antibody-based therapies.

Weaknesses:

A His tagged antigen was used for immunization and H1-his was used in all assays. Either the removal of His specific clones needs to be done before selection, or a different tag needs to be used in the subsequent assays.

This assay doesn't directly test the neutralization of influenza but rather equates viral clearance to competitive inhibition. The results would be strengthened with the demonstration of a functional antibody in vivo with viral clearance.

Limitations of this new technique are as follows: there is a significant loss of cells during FACs, transfection and cloning efficiency are critical to success, and well-based systems limit the number of possible clones (as the author discussed in the conclusions). Early enrichment of the B cells could improve efficiency, such as selection for memory B cells.