Our studies in FY2016 have addressed several major questions: 1. Three years ago we established a CRADA with Boehringer-Ingelheim Pharmaceuticals to generate a panel of mAbs targeting expanded hTregs. Our initial strategy was to immunize mice with hTregs expanded in culture for 14 days, and then screen the resulting hybridomas for binding to expanded hTregs, but not to activated Tconv cells. We subsequently altered our strategy to minimize responses to non-Treg antigens by immunizing first with HeLa cells, followed by cyclophosphamide treatment to deplete responding B cells. Supernatants from hybridomas generated by this protocol were screened by flow cytometry for preferential binding to expanded Treg cells compared to expanded Teff cells and HeLa cells. Hybridomas were also tested in vitro in T cell suppression assays to look for modulation of Treg function. mAbs were also used in western blot analysis to identify target antigen sizes. mAbs that showed binding in western blots, were also tested for their ability to immunoprecipitate their target antigen. We grouped mAbs together based on staining patterns, antigen size as determined by western blot analysis or as unique antibodies. One large group of mAbs identified an epitope on CD25 that was expressed at high levels on expanded Treg, but at much lower levels on expanded Teff cells. These unique anti-CD25 mAbs did not inhibit T cell proliferation, modulate Treg suppression, or block IL-2 signaling (proliferation or STAT5 phosphorylation). We currently have 12 additional candidate mAbs that require further characterization to identify their target antigens. These mAbs have failed to reproducibly bind antigen in western blot analysis and we have been unable to immunoprecipitate their target antigens for mass spec analysis. To circumvent this difficulty, we have submitted these antibodies for a surface receptor microarray screen carried out by Retrogenix. Retrogenix has a library of over 4500 human membrane proteins individually over-expressed in human cells. Our mAbs are now being screened for binding to this library and a number of target antigens have been identified. We are currently in the process of obtaining expression vectors for the antigens identified in our screen to confirm target antigen identity. 2. One of the major problems in the preparation of Tregs for cellular biotherapy is the stability of Foxp3 expression during the expansion cultures. We have demonstrated that the addition of oligonucleotides tp the expansion cultures stabilizes the expression of Foxp3 and Helios. We have dissected the molecular basis for the enhancement of Foxp3 stability during hTreg expansion by the addition of phosphorothioated oligonucleotides (ODNps25). We identified SAMHD1 (sterile alpha motif and HD domain 1) as an ODNps25 binding protein and demonstrated that ODNs interact with SAMHD1 to interfere with its nuclease activity, thereby stabilizing the 3' UTR of FoxP3 mRNA in long-term culture.
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