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

Abstract

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. To demonstrate this notion, a murine myeloid progenitor cell line 32Dcl3, which proliferates in an IL-3-dependent manner and differentiates into granulocytes when cultured in the presence of G-CSF, is chosen as a model. To mimic the cell fate control of 32Dcl3 cells, IL-3R-based piCAR and G-CSFR-based diCAR are rationally designed and co-expressed in 32Dcl3 cells to evaluate the proliferation- and differentiation-inducing functions. Consequently, the sequential induction from proliferation to differentiation with switching the cytokine from IL-3 to G-CSF is successfully replaced by switching the antigen from one to another in the CARs-co-expressing cells. Thus, piCAR and diCAR may become a platform technology for sequentially controlling proliferation and differentiation of various cell types that need to be produced in cell and gene therapies.

Phenotypic screening of signaling motifs that efficiently induce cell proliferation

“…To further verify the applicability of this screening system, we employed a myeloid progenitor cell line 32Dcl3, which not only proliferate in response to IL-3 but also differentiate into granulocytes when stimulated with G-CSF 27 , 28 . Unlike Ba/F3 cells, 32Dcl3 cells may have a narrower window for cell proliferation because different cytokine signals induce different cell fates (Fig.…”

Harnessing bioengineered myeloid progenitors for precision immunotherapies

“…The introduction of proliferation- and differentiation-inducing CARs 41 may furthermore allow for self-regulating systems that produce effector cells in vivo; for only as long as a certain target antigen is present 41 . Potential risks of therapy persistence or malignant transformation could be mitigated by transfecting proliferation-inducing CAR-myeloid cells via non-integrating mRNA or by introducing ‘safety switches’ to improve product safety 42 .…”