Anton Simeonov Ph.D. National Institute of Health
In this literature review, learn how researchers converted stem cells to dendritic cells using agonist antibodies.
The work by Yea and team* further extends the infective antibody library platform developed by the Lerner team (see commentary: Assay Drug Dev Technol 2013;11:347): in it, antibody libraries are incorporated into lentiviral vectors allowing linking of antibody genotype and phenotype, with the host cell becoming a de facto reporter for the antibody property. While the earlier work utilized a library enriched for binders through a preselection panning step and utilized a reporter to detect a functional outcome, here the system is pushed to the limit by using a naïve library and direct observational selection.
A 108-member naïve human antibody library incorporated into lentiviral vectors was used to infect TF-1 erythroblast cells contained within methylcellulose agar substrate (Figures A, B, and C). Colonies produced in this manner exhibited a range of morphologies that could be singled out through simple direct observation. The authors studied the cellular background of the colonies with the most unusual morphology and identified three antibody clones whose target was determined through mass spectrometry to be primarily the integrin receptor. The antibodies acted as agonists, activating downstream pathways and were further shown to induce human bone marrow CD34+ stem cells to differentiate into cells of the macrophage lineage to form dendritic cells.
Scheme for the unbiased selection of active antibodies against cellular targets from combinatorial libraries. Figure A: Lentiviral antibody libraries were constructed from naïve human combinatorial antibody libraries. Plasmids of lentiviral antibody libraries and virus packaging were used to transiently transfect HEK293T cells. TF-1 erythroblast cells were infected with lentiviral antibody libraries. The resulting cells were grown on methylcellulose agar for morphogenic screening. Thirty colonies that showed unusual and different morphologies were picked after 2 weeks and antibody genes were recovered by PCR. After verification of the activity of the antibodies by the MTS cell proliferation assay, each antibody was incubated with cell lysates followed by collection of the complexes on protein A/G beads. The beads were collected and washed, and the bound proteins were eluted by acid (pH 2.8) and separated on SDS/PAGE gels that were silver-stained. The bands of gel were sliced and analyzed by nano-LC-MS/MS after trypsinization.
Figure B: Representative morphologies of colonies. Figure C: All possible combinations of antibody genes isolated from the same colonies were transfected into HEK293T cells. TF-1 cell proliferation was tested using the conditioned medium from HEK293T cells 48 h after transfection of antibodies. PCR, polymerase chain reaction; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; SDS/PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; LC-MS/MS, liquid chromatography with tandem mass spectrometry; GM-CSF, granulocyte macrophage colony-stimulating factor.
Additionally, the authors point out that the antibody’s differentiation mechanisms is distinct from that caused by the granulocyte macrophage colony-stimulating factor in which phosphorylation of the key transcription factor, STAT5, is involved. Thus, the use of infective antibody libraries in combination with a simple selection based on morphology change resulted in the identification of rare agonist antibodies that caused a robust stem cell differentiation. Unlike other antibody selection schemes that are typically based on identification of superior binders, here the evolutionary process of testing many antibodies for a functional outcome is manifested through an acquisition of a fitness trait; that is, the growth stimulation of a colony of unusual morphology.
*Abstract from PNAS USA 2013, Volume 110: 14966–14971
When combinatorial antibody libraries are rendered infectious for eukaryotic cells, the integrated antibody genotype and cellular phenotype become permanently linked and each cell becomes a selection system unto itself. These systems should be ideal for the identification of proteins and pathways that regulate differentiation so long as selection systems can be devised. Here we use a selection system based on the ability of secreted antibodies to alter the morphology of colonies expressing them when grown in soft agar.
Importantly, this approach is different from all previous studies in that it used a pure discovery format where unbiased libraries that were not preselected against any known protein were used as probes. As such, the strategy is analogous to classical forward genetic approaches except that it operates directly at the protein level. This approach led to the identification of integrin binding agonist antibodies that efficiently converted human stem cells to dendritic cells.
Anton Simeonov, Ph.D., works at the NIH.
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Proceedings of the National Academy of Sciences USA titled “Converting stem cells to dendritic cells by agonist antibodies from unbiased morphogenic selections.” Authors of the paper are Yea K, Zhang H, Xie J, Jones TM, Yang G, Song BD, and Lerner RA.