Sequential control of myeloid cell proliferation and differentiation by cytokine receptor-based chimeric antigen receptors.

As chimeric antigen receptor (CAR)-T cell therapy has been recently applied in clinics, controlling the fate of blood cells is increasingly important for curing blood disorders. In this study, we aim to construct proliferation-inducing and differentiation-inducing CARs (piCAR and diCAR) with two different antigen specificities and express them simultaneously on the cell surface. Since the two antigens are non-cross-reactive and exclusively activate piCAR or diCAR, sequential induction from cell proliferation to differentiation could be controlled by switching the antigens added in the culture medium.

Cell fate-inducing CARs orthogonally control multiple signaling pathways.

While chimeric antigen receptor (CAR) has been clinically applied in T cell-mediated cancer therapy, CARs that combinatorially control general cell fates have yet to be practical. In this study, we aim to control multiple cell fates simultaneously by combinatorial activation of multiple cell fate-inducing CARs (cfiCARs). We construct CARs that transduce two different signals using two antigen-specific CARs.

Cell-Surface Expression Levels Are Important for Fine-Tuning the Performance of Receptor Tyrosine Kinase-Based Signalobodies.

As receptor tyrosine kinases (RTKs) play important roles in cell-fate control of various cell types, engineered RTKs that could respond to inexpensive ligands might drastically reduce the cost of producing desired cells for various applications in regenerative medicine. We developed several engineered RTKs named "signalobodies" in which the ligand-recognition domain of RTKs is replaced by single-chain Fv for enabling recognition of a specific antigen. However, the remaining concern was the dysregulation of antigen-dependent on/off signaling of the signalobodies.

Differentiation signalobody: Demonstration of antigen-dependent osteoclast differentiation from a progenitor cell line.

A "cytokine-less" in vitro differentiation method would be promising for cost-effective mass production of cells used for regenerative medicine. In this study, we developed a differentiation signalobody S-RANK, in which the extracellular domain of receptor activator of nuclear factor kappa-B (RANK) is replaced with a single-chain variable fragment (scFv) to attain signaling in response to an inexpensive antigen. A murine macrophage cell line RAW264, which is known to differentiate into an osteoclast by RANK ligand (RANKL), was lentivirally transduced with S-RANK.

Construction of antibody/insulin receptor chimera for growth induction of mammalian cells.

The insulin receptor (IR) is expressed ubiquitously in various tissues, where insulin exerts various biological effects on the target cells, such as cellular metabolic changes, cell proliferation and differentiation. Therefore, mimicry of insulin signaling would be a promising strategy to realize artificial control of such cellular fates. In this study, we constructed an antibody/insulin receptor chimera that enables to utilize any antigen as the ligand in principle.