The use of allogeneic, pluripotent stem-cell-derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator-initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)-homozygous-induced pluripotent stem cell (iPSC)-derived antiglypican-3 (GPC3) chimeric antigen receptor (CAR)-expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder-free production of CAR-expressing NK/ILC cells from CAR-transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8-3.6 × 10 6 iPSC within 7 weeks was 1.8-4.0 × 10 9 . These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN-γ) production against GPC3-expressing tumor cells. When the CAR-NK/ILC cells were injected into a GPC3-positive, ovarian-tumor-bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non-clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR-NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell-based immune cell cancer therapies. K E Y W O R D Schimeric antigen receptor, GPC3, ILC, immunotherapy, iPSC, NK | 1479 UEDA Et Al.

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubuleassociated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factortreated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp4O or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp4O. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.

SummaryNerve growth factor (NGF)-and epidermal growth factor (EGF)-stimulated MAP kinase activities from PC-12 cells were recently found to be resolvable into two peaks (microtubule-associated protein[MAP] kinases I and II) by ion-exchange or hydrophobic-interaction HPLC, and both kinases I and II were activated by various growth factors, by okadaic acid, and by phorbol esters. In the present study both MAP kinases I and II were also activated by bombesin/gastrin-releasing peptide (GRP) in Swiss 3T3 cells, and the peak of activation in response to GRP occurred earlier than that observed with EGF. In this work, we confirmed the results reported by Bierman et al. (J. Cell. Phys., 142,[441][442][443][444][445][446][447][448]. Since the cDNA for the bombesin/GRP receptor indicates that this receptor has a transmembrane topology like that of other G-proteincoupled receptors, an alternative signal transduction pathway may mediate the activation of MAP kinase by bombesin/GRP. Key Words: bombesin/GRP, MAP kinase, Swiss 3T3, nerve growth factor, epidermal growth factor Bombesin/gastrin-releasing peptide (GRP) and epidermal growth factor (EGF) receptors exert their mitogenic effects in Swiss 3T3 cells by distinct pathways [1]. A cDNA for the bombesin/GRP receptor has been cloned [2,3], and the protein has been shown to have a transmembrane topology like that of other G protein-coupled receptors. Although tyrosine kinase activity has been associated with the receptor function as reported by Isacke et al . [4], there is no tyrosine kinase homology in the receptor sequence. To evaluate the molecular

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