Chihaya Imai

Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia

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 CD8α, and the signaling domain of CD3ζ. We compared the antileukemic activity mediated by a novel receptor (‘anti-CD19-BB-ζ’) 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-ζ receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-ζ 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-ζ-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+ B-lymphoid malignancies.

Expansion of Highly Cytotoxic Human Natural Killer Cells for Cancer Cell Therapy

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. Seven-day 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.

Development and functional characterization of human bone marrow mesenchymal cells immortalized by enforced expression of telomerase

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.

T Lymphocytes Expressing a CD16 Signaling Receptor Exert Antibody-Dependent Cancer Cell Killing

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, CD8α hinge, and transmembrane domains, along with signaling domains from CD3ζ and 4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-ζ. After retrovirus-mediated expression in human T cells, CD16V-BB-ζ bound humanized antibodies with higher affinity than a control receptor containing the more common F158 variant. Engagement of CD16V-BB-ζ 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-ζ 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-ζ-mediated killing of HER2 (ERBB2)(+) breast and gastric cancer cells; similar results were obtained with an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration of CD16V-BB-ζ T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo. Signaling mediated by 4-1BB-CD3ζ induced higher T-cell activation, proliferation, and cytotoxicity than CD3ζ or FcεRIγ, 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-ζ as a universal, next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer.

Replicative potential of human natural killer cells.

The 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 1·6 × 105 to 1·2 × 1011% (median, 5·9 × 106%; 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.

Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15.

Natural killer (NK) cell survival and, hence, cytotoxicity requires cytokine support. We determined whether expression of interleukin-15 (IL-15) in a nonsecretory, membrane-bound form could sustain NK cell growth. We linked the human IL15 gene to that encoding CD8α transmembrane domain (mbIL15). After retroviral transduction, human NK cells expressed mbIL15 on the cell surface; IL-15 secretion was negligible. Survival of mbIL15-NK cells without interleukin-2 (IL-2) after 7-day culture was vastly superior to that of mock-transduced NK cells (P < .001, n = 15) and of NK cells expressing nonmembrane-bound IL-15 (P = .025, n = 9); viable mbIL15-NK cells were detectable for up to 2 months. In immunodeficient mice, mbIL15-NK cells expanded without IL-2 and were detectable in all tissues examined (except brain) in much higher numbers than mock-transduced NK cells (P < .001). Expansion further increased with IL-2. The primary mechanism of mbIL15 stimulation was autocrine; it activated IL-15 signaling and antiapoptotic signaling. NK cells expressing mbIL15 had higher cytotoxicity against leukemia, lymphoma, and solid tumor cells in vitro and against leukemia and sarcoma cells in xenograft models. Thus, mbIL15 confers independent growth to NK cells and enhances their antitumor capacity. Infusion of mbIL15-NK cells would allow NK cell therapy without the potential adverse effects of cytokine administration.

Synergistic and persistent effect of T-cell immunotherapy with anti-CD19 or anti-CD38 chimeric receptor in conjunction with rituximab on B-cell non-Hodgkin lymphoma

Using artificial receptors, it is possible to redirect the specificity of immune cells to tumour‐associated antigens, which is expected to provide a useful strategy for cancer immunotherapy. Given that B‐cell non‐Hodgkin lymphoma (B‐NHL) cells invariably express CD19 and CD38, these antigens may be suitable molecular candidates for such immunotherapy. We transduced human peripheral T cells or a T‐cell line with either anti‐CD19‐chimeric receptor (CAR) or anti‐CD38‐CAR, which contained an anti‐CD19 or anti‐CD38 antibody‐derived single‐chain variable domain respectively. Retroviral transduction led to anti‐CD19‐CAR or anti‐CD38‐CAR expression in T cells with high efficiency (>60%). The T cell line, Hut78, when transduced with anti‐CD19‐CAR or anti‐CD38‐CAR, exerted strong cytotoxicity against the B‐NHL cell lines, HT and RL, and lymphoma cells isolated from patients. Interestingly, use of both CARs had an additive cytotoxic effect on HT cells in vitro. In conjunction with rituximab, human peripheral T cells expressing either anti‐CD19‐CAR or anti‐CD38‐CAR enhanced cytotoxicity against HT‐luciferase cells in xenografted mice. Moreover, the synergistic tumour‐suppressing activity was persistent in vivo for over 2 months. These results provide a powerful rationale for clinical testing of the combination of rituximab with autologous T cells carrying either CAR on aggressive or relapsed B‐NHLs.

Activated T-cell-mediated immunotherapy with a chimeric receptor against CD38 in B-cell non-Hodgkin lymphoma.

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 non-Hodgkin 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 vector-transduced 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.

4-1BB chimeric antigen receptors.

AbstractIn addition to T-cell receptor signals, T lymphocytes require costimulatory signals for robust activation. Among these, those mediated by 4-1BB (CD137, TNFRSF9) are critical for tumor immunity. 4-1BB is expressed in T-cell receptor–activated lymphocytes as well as natural killer cells and other hematopoietic and nonhematopoietic cells. 4-1BB ligation induces a signaling cascade that results in cytokine production, expression of antiapoptotic molecules, and enhanced immune responses. In line with the described function of 4-1BB, its addition to CD3&zgr; chimeric antigen receptors (CARs) increases their capacity to provoke T-cell expansion and antitumor activity. The results of preclinical studies with 4-1BB CARs have been corroborated by encouraging results from clinical trials. Advantages and disadvantages of 4-1BB CARs versus CARs bearing other costimulatory components remain to be fully elucidated. In this review, we discuss the properties of 4-1BB, the design of 4-1BB CARs, and the function of T lymphocytes and natural killer cells expressing them.

Characterization of pediatric Philadelphia-negative B-cell precursor acute lymphoblastic leukemia with kinase fusions in Japan

Recent studies revealed that a substantial proportion of patients with high-risk B-cell precursor acute lymphoblastic leukemia (BCP-ALL) harbor fusions involving tyrosine kinase and cytokine receptors, such as ABL1, PDGFRB, JAK2 and CRLF2, which are targeted by tyrosine kinase inhibitors (TKIs). In the present study, transcriptome analysis or multiplex reverse transcriptase–PCR analysis of 373 BCP-ALL patients without recurrent genetic abnormalities identified 29 patients with kinase fusions. Clinically, male predominance (male/female: 22/7), older age at onset (mean age at onset: 8.8 years) and a high white blood cell count at diagnosis (mean: 94 200/μl) reflected the predominance of National Cancer Institute high-risk (NCI-HR) patients (NCI-standard risk/HR: 8/21). Genetic analysis identified three patients with ABL1 rearrangements, eight with PDGFRB rearrangements, two with JAK2 rearrangements, three with IgH-EPOR and one with NCOR1-LYN. Of the 14 patients with CRLF2 rearrangements, two harbored IgH-EPOR and PDGFRB rearrangements. IKZF1 deletion was present in 16 of the 22 patients. The 5-year event-free and overall survival rates were 48.6±9.7% and 73.5±8.6%, respectively. The outcome was not satisfactory without sophisticated minimal residual disease-based stratification. Furthermore, the efficacy of TKIs combined with conventional chemotherapy without allogeneic hematopoietic stem cell transplantation in this cohort should be determined.