Summary Investigators at the University of Minnesota have teamed up to engineer novel protein activators of the Notch family of cell surface receptors, which are master regulators of T-cell differentiation from induced pluripotent stem cells. This technology will accelerate the development of engineered T cell therapies for treating cancer as well as a range of diseases including auto-immune disorders, infectious disease, immune-deficiencies, graft vs. host disease, and organ transplant rejection. Existing FDA approved engineered T cells are powerful therapeutics yet major challenges remain, including difficulty in differentiating T cells from precursor cell types and difficulty in editing and validating precursor cells prior to differentiation. To overcome these limitations and to enable a transformative jump in T cell engineering approaches, the research team is developing reagents that target and trigger conformational opening of the proteolytic switch NRR domain of Notch1. The Notch NRR buries a cryptic protease site that is normally only exposed to its protease physiologically by tugging forces generated during binding of its ligand on a neighboring cell. The project aims to develop soluble nanobody reagents that functionally pry open the domain and remove the requirement for co-culture with Notch ligands during T cell differentiation. In Aim 1 of the project, phage-display protein engineering is used to identify and optimize nanobodies that selectively bind structurally distinct states of the Notch1 NRR. In Aim 2, these nanobodies are strategically linked together in arrays for creating potent Notch1 activators by inducing conformational “opening” of the NRR. In Aim 3, iPSC cell lines that report Notch1 signaling and T cell commitment will be developed, characterized, and then used in assays monitoring differentiation of iPSCs into T cells under induction by engineered Notch1 activators. In the final stage of the project, the reporter cell lines will be used in benchmarking experiments comparing the performance of Notch1 activators developed in Aim 1 and 2, to industry standard technologies. The milestones of this project are: (a) generating a tool set of molecules that bind selectively to the Notch1 NRR; (b) developing a panel of iPSC reporter systems that monitor commitment and differentiation of iPSCs to T cells that have normal physiologic function including cell killing potential; (c) creating potent and selective Notch1 activators that induce iPSCs to differentiate into T cells with a marked improvement in the efficiency differentiation and expansion over current approaches. This will enable development of improved cancer treatments, and improve health disparities by increasing access to treatment with a standardized iPSC-based cell source that can be frozen and banked for multiple doses while significantly bringing down cost per product.

Project Narrative This project merges expertise in structural biology, protein engineering, T cell biology, and oncology to engineer soluble nanobody-based Notch1 activators that will transform and accelerate the development of novel T cell-based therapies to treat cancer.