To develop a therapy for drug-resistant B-lineage acute lymphoblastic leukemia (ALL), we transduced T lymphocytes with anti-CD19 chimeric receptors, consisting of an anti-CD19 single-chain variable domain (reactive with most ALL cases), the hinge and transmembrane domains of CD8a, and the signaling domain of CD3f. We compared the antileukemic activity mediated by a novel receptor ('anti-CD19-BB-f') containing the signaling domain of 4-1BB (CD137; a crucial molecule for T-cell antitumor activity) to that of a receptor lacking costimulatory molecules. Retroviral transduction produced efficient and durable receptor expression in human T cells. Lymphocytes expressing anti-CD19-BB-f receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-f lymphocytes were remarkably effective in cocultures with bone marrow mesenchymal cells, and against leukemic cells from patients with drug-resistant ALL: as few as 1% anti-CD19-BB-f-transduced T cells eliminated most ALL cells within 5 days. These cells also expanded and produced interleukin-2 in response to ALL cells at much higher rates than those of lymphocytes expressing equivalent receptors lacking 4-1BB. We conclude that anti-CD19 chimeric receptors containing 4-1BB are a powerful new tool for T-cell therapy of B-lineage ALL and other CD19 þ B-lymphoid malignancies.

IntroductionB-cell malignancies of children and adults, such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL), are often incurable even with intensive chemotherapy. For many patients, bone marrow ablation followed by allogeneic hematopoietic stem cell transplantation is the only potentially curative option, but the disease may return after transplantation. 1 The well-documented association between T-cellmediated graft-versus-host disease (GvHD) and a delay or suppression of leukemic relapse after allogeneic stem cell transplantation 2-4 has led some investigators to manipulate GvHD by infusion of donor T lymphocytes. Although this procedure can induce a measurable antineoplastic response, [5][6][7][8] it carries the risk of severe GvHD, particularly in those patients (Ͼ 70%) who lack an HLA-identical donor. Moreover, in some B-cell malignancies, such as ALL, the effect of lymphocyte infusions is often inadequate. 6,9,10 Besides T lymphocytes, natural killer (NK) cells also exert cytotoxicity against cancer cells. 11 Recent studies have emphasized the potential of NK-cell therapy in recipients of allogeneic hematopoietic stem cell transplants. In animal models of transplantation, donor NK cells could lyse leukemic cells and host lymphohematopoietic cells without affecting nonhematopoietic tissues, 12 suggesting that NK-mediated graft-versus-leukemia responses may occur in the absence of systemic disease. Because NK cells are inhibited by self-HLA molecules, which bind to killer immunoglobulin-like receptors (KIRs), these findings have led to the clinical practice of selecting hematopoietic stem cell transplantation donors with an HLA and KIR type that favors NK-cell activation and thus could be expected to promote an antileukemic effect. [13][14][15] However, selection of the "best" donor is limited to patients who have more than one potential donor and the capacity of NK cells to lyse lymphoid cells is generally low and difficult to predict. 13,[15][16][17] Emerging evidence indicates that T lymphocytes genetically modified with chimeric receptors able to recognize a surface molecule of target cells and transduce activation signals can specifically enhance T-cell cytotoxicity against cancer cells both in vitro and in vivo. [18][19][20][21] The studies presented here are based on the concept that expression of chimeric receptors on NK cells could overcome HLA-mediated inhibitory signals, thus endowing the cells with cytotoxicity against otherwise NK-resistant cells. To test this hypothesis, we first developed a novel method that allows specific and vigorous expansion of NK cells lacking T-cell receptors (CD56 ϩ CD3 Ϫ cells) and their highly efficient transduction with chimeric receptors. Then, we tested the relative antileukemic effects of genetically modified NK cells bearing chimeric receptors (directed against CD19, a molecule widely expressed by malignant B cells) that deliver different primary and costimulatory signals. Materials and methods CellsThe C...

Infusions of natural killer (NK) cells are an emerging tool for cancer immunotherapy. The development of clinically applicable methods to produce large numbers of fully functional NK cells is a critical step to maximize the potential of this approach. We determined the capacity of the leukemia cell line K562 modified to express a membrane-bound form of interleukin (IL)-15 and 41BB ligand (K562-mb15-41BBL) to generate human NK cells with enhanced cytotoxicity. Sevenday coculture with irradiated K562-mb15-41BBL induced a median 21.6-fold expansion of CD56 + CD3 -NK cells from peripheral blood (range, 5.1-to 86.6-fold; n = 50), which was considerably superior to that produced by stimulation with IL-2, IL-12, IL-15, and/or IL-21 and caused no proliferation of CD3 + lymphocytes. Similar expansions could also be obtained from the peripheral blood of patients with acute leukemia undergoing therapy (n = 11). Comparisons of the gene expression profiles of the expanded NK cells and their unstimulated or IL-2-stimulated counterparts showed marked differences. The expanded NK cells were significantly more potent than unstimulated or IL-2-stimulated NK cells against acute myeloid leukemia cells in vitro. They could be detected for >1 month when injected into immunodeficient mice and could eradicate leukemia in murine models of acute myeloid leukemia. We therefore adapted the K562-mb15-41BBL stimulation method to large-scale clinical-grade conditions, generating large numbers of highly cytotoxic NK cells. The results that we report here provide rationale and practical platform for clinical testing of expanded and activated NK cells for cell therapy of cancer. [Cancer Res 2009;69(9):4010-7]

To expand applications for T-cell-based immunotherapy in cancer, we designed a receptor that binds the Fc portion of human immunoglobulins and delivers activation signals. The construct included the high-affinity CD16 (FCGR3A) V158 variant, CD8a hinge, and transmembrane domains, along with signaling domains from CD3z and 4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-z. After retrovirus-mediated expression in human T cells, CD16V-BB-z bound humanized antibodies with higher affinity than a control receptor containing the more common F158 variant. Engagement of CD16V-BB-z provoked T-cell activation, exocytosis of lytic granules, and sustained proliferation, with a mean cell recovery after 4-week coculture with Daudi lymphoma cells and rituximab of nearly 70-fold relative to input cells. In contrast, unbound antibody alone produced no effect. CD16V-BB-z T cells specifically killed lymphoma cells and primary chronic lymphocytic leukemia cells in combination with rituximab at a low effector:target ratio, even when assayed on mesenchymal cells. Trastuzumab triggered CD16V-BB-z-mediated killing of HER2 (ERBB2) þ breast and gastric cancer cells; similar results were obtained with an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration of CD16V-BB-z T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo.Signaling mediated by 4-1BB-CD3z induced higher T-cell activation, proliferation, and cytotoxicity than CD3z or FceRIg, and the receptor was expressed effectively after mRNA electroporation without viral vectors, facilitating clinical translation. Our results offer preclinical proof of concept for CD16V-BB-z as a universal, next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer. Cancer Res; 74(1); 93-103. Ó2013 AACR.

Summary. To create immortal mesenchymal cell lines, we transduced primary human bone marrow mesenchymal cells with telomerase reverse transcriptase (TERT). TERT + mesenchymal cells continued to grow for > 2 years; parallel TERT -cultures underwent senescence after 15 weeks. TERT + mesenchymal cells did not form foci in soft agar, had a normal karyotype and could differentiate into osteoblasts and chondrocytes. Their capacity to support leukaemic lymphoblasts and normal CD34 + haematopoietic cells was equal to or greater than that of primary cells; 42 TERT + mesenchymal cell clones varied in their supporting capacity. Immortalized mesenchymal cells offer a promising tool for identifying molecules that regulate human haematopoiesis.

Key Points Expression of IL-15 in a membrane-bound form sustains NK cell survival and expansion in vitro and in vivo without exogenous cytokines. These NK cells have superior cytotoxicity against leukemia, lymphoma, and solid tumor cells, supporting their clinical testing.

SummaryThe replicative potential of human CD56 + CD3 ) natural killer (NK) cells is unknown. We found that by exposing NK cells to the leukaemic cell line K562 genetically modified to express 4-1BB ligand and interleukin 15 (K562-mb15-41BBL), they expanded for up to 30 population doublings, achieving numbers that ranged from 1AE6 · 10 5 to 1AE2 · 10 11 % (median, 5AE9 · 10 6 %; n = 7) of those originally seeded. However, NK cells eventually became unresponsive to stimulation and died. Their demise could be suppressed by enforcing the expression of the human telomerase reverse transcriptase gene (TERT). TERT-overexpressing NK cells continued to proliferate in response to K562-mb15-41BBL stimulation for more than 1 year of culture, while maintaining a normal karyotype and genotype. Long-lived NK cells had high cytotoxicity against myeloid and T-lineage leukaemic cells. They remained susceptible to genetic manipulation, becoming highly cytotoxic to B-lineage leukaemic cells after expression of anti-CD19 signaling receptors. Thus, human NK cells have a replicative potential similar to that of T lymphocytes and their lifespan can be significantly prolonged by an increase in TERT activity. We suggest that the methods described here should have many applications in studies of NK cell biology and NK cell-based therapies.

T-cell-mediated immunotherapy with a chimeric antigen receptor (CAR) is expected to become a powerful treatment for cancer. CD38, highly expressed in B-cell nonHodgkin lymphoma (B-NHL) cells, is an attractive target in immunotherapy for B-NHL. We retrovirally transduced a T-cell line, Hut78, expressing little CD38, with an anti-CD38-CAR. Hut78 cells with the anti-CD38-CAR were cocultured with B-NHL cell lines bearing CD38 and also B-NHL cells from patients. Four days later most of the lymphoma cells were killed (the level of cytotoxicity was >95%). By contrast, there was undetectable cytotoxicity against CD38-negative cell lines. Then, we introduced the anti-CD38-CAR into human peripheral T cells. However, the recovery of viable cells was very low, presumably because of an autolytic reaction caused by the association of the anti-CD38-CAR with CD38 on the cell surface. The addition of an anti-CD38 antibody increased the yield of viable transduced T cell probably by blocking the autolytic reaction. We cocultured human peripheral T cells bearing anti-CD38-CAR with B-NHL cells. The median specific cytotoxicity was greater than 90%. These cells were injected 4 times into NOD/SCID mice, which were inoculated with B-NHL cells luciferase. Luciferase activity was not detectable even 30 days after the inoculation in 5 of 6 mice injected. By contrast, it increased in all of the mice injected with the mock vectortransduced T cell. In conclusion, T cell with the anti-CD38-CAR showed powerful cytotoxicity against B-NHL cells in vitro and in vivo. These findings may provide an important clue for improving the methodology of T-cell-mediated immunotherapy.