Key Points GD2-specific CAR renders NKT cells cytotoxic against NB cells and results in potent in vivo antitumor activity without graft-versus-host disease. The 4-1BB-containing CAR constructs induce T helper 1–like polarization in NKT cells.

Vα24-invariant NKT cells inhibit tumor growth by targeting tumor-associated macrophages (TAMs).

NOTCH1 is activated by mutation in more than 50% of human T-cell acute lymphoblastic leukemias (T-ALLs) and inhibition of Notch signaling causes cell-cycle/ growth arrest, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor phosphatase and tensin homolog (PTEN) is also mutated or lost in up to 20% of cases. It was recently observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition, raising concern that patients with PTEN-negative disease may fail Notch inhibitor therapy. As these studies were limited to established cell lines, we addressed this issue using a genetically defined mouse retroviral transduction/bone marrow transplantation model and observed primary murine leukemias to remain dependent on NOTCH1 signaling despite Pten loss, with or without additional deletion of p16 Ink4a /p19 Arf IntroductionThe 4 mammalian Notch genes (NOTCH1-4) encode a family of highly conserved type I transmembrane receptors that are normally activated by ligands of the Delta/Serrate/Lag-2 family expressed on the surface of neighboring cells. Once activated by ligand, the Notch receptors undergo proteolytic cleavage first by a disintegrin and metalloprotease, then by a ␥-secretase, which releases the intracellular domain (ICN) from the plasma membrane to translocate to the nucleus to stimulate transcription of downstream target genes in complex with the DNA-binding factor CBF1(RBPJ)/ suppressor of hairless/Lag-1 and coactivators of the Mastermind family. Although regulated NOTCH1 signaling is important for normal T-cell development, 1 it is frequently activated by mutation in the human cancer T-cell acute lymphoblastic leukemia (T-ALL). 2 The potent oncogenicity of activated NOTCH1 has been demonstrated in murine bone marrow transduction/transplantation models and several transgenic mouse lines. 3 Activating NOTCH1 mutations occur in more than 50% of primary human T-ALLs and cluster in the heterodimerization (HD) and C-terminal proline-, glutamic acid-, serine-, and threonine-rich (PEST) domains. 4 HD mutations result in weakened association or complete dissociation of the receptor subunits, and thus lead to heightened/constitutive activation of the receptor. 5 PEST domain mutations often generate premature stop codons that delete the PEST degron, and thus enhance signaling by reducing turnover/ prolonging half-life of activated ICN. 6 When present together, the HD and PEST mutations occur in cis, and stimulate signaling in a synergistic fashion. 4 Interestingly, a similar overall frequency of Notch1 mutations (mostly PEST, but some HD) has been observed in various mouse models of T-ALL, underscoring the importance of Notch1 signaling in T-cell leukemogenesis. In addition, both human and murine T-ALL cells bearing NOTCH1 mutations are frequently sensitive to treatment with inhibitors of Notch signaling including ␥-secretase inhibitors (GSIs) that induce G1 cell-cycle/ growth arrest and in some cases apoptosis. 4,7-10 Based on these findings, GSIs and other inhi...

T cells engineered to express CD19-specific chimeric antigen receptors (CARs) have shown breakthrough clinical successes in patients with B-cell lymphoid malignancies. However, similar therapeutic efficacy of CAR T cells in solid tumors is yet to be achieved. In this study we systematically evaluated a series of CAR constructs targeting glypican-3 (GPC3), which is selectively expressed on several solid tumors. We compared GPC3-specific CARs that encoded CD3ζ (Gz) alone or with costimulatory domains derived from CD28 (G28z), 4-1BB (GBBz), or CD28 and 4-1BB (G28BBz). All GPC3-CARs rendered T cells highly cytotoxic to GPC3-positive hepatocellular carcinoma, hepatoblastoma, and malignant rhabdoid tumor cell lines in vitro. GBBz induced the preferential production of Th1 cytokines (interferon γ/granulocyte macrophage colony-stimulating factor) while G28z preferentially induced Th2 cytokines (interleukin-4/interleukin-10). Inclusion of 4-1BB in G28BBz could only partially ameliorate the Th2-polarizing effect of CD28. 4-1BB induced superior expansion of CAR T cells in vitro and in vivo. T cells expressing GPC3-CARs incorporating CD28, 4-1BB, or both induced sustained tumor regressions in two xenogeneic tumor models. Thus, GBBz CAR endows T cells with superior proliferative potential, potent antitumor activity, and a Th1-biased cytokine profile, justifying further clinical development of GBBz CAR for immunotherapy of GPC3-positive solid tumors.